Human G-protein chemokine receptor (CCR5) HDGNR10

ABSTRACT

The present invention relates to a novel human protein called Human G-protein Chemokine Receptor (CCR5) HDGNR10, and isolated polynucleotides encoding this protein. The invention is also directed to human antibodies that bind Human G-protein Chemokine Receptor (CCR5) HDGNR10 and to polynucleotides encoding those antibodies. Also provided are vectors, host cells, antibodies, and recombinant methods for producing Human G-protein Chemokine Receptor (CCR5) HDGNR10 and human anti-Human G-protein Chemokine Receptor (CCR5) HDGNR10 antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to this novel human protein and these novel human antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/067,800, filed 8 Feb. 2002, now U.S. Pat. No. 7,175,988; and claimsthe benefit of U.S. Provisional Application No. 60/341,725, filed 21Dec. 2001; each of said applications is hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the G-protein Chemokine Receptor (CCR5)family. More specifically, the present invention relates to apolynucleotide encoding a novel human polypeptide named Human G-proteinChemokine Receptor (CCR5) HDGNR10, referred to herein as “G-proteinChemokine Receptor” or “HDGNR10.” This invention also relates toG-protein Chemokine Receptor (CCR5) polypeptides, as well as vectors,host cells, antibodies directed to G-protein Chemokine Receptor (CCR5)polypeptides, and the recombinant methods for producing the same. Alsoprovided are diagnostic methods for detecting diseases, disorders,and/or conditions related to the immune system and HIV infection, andtherapeutic methods for treating, preventing, and/or diagnosing suchdiseases, disorders, and/or conditions. The invention further relates toscreening methods for identifying agonists and antagonists of G-proteinChemokine Receptor (CCR5) activity. The G-protein Chemokine Receptor(CCR5) is also known as CCR5.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins orPPG-proteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656(1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteinsthemselves, effector proteins, e.g., phospholipase C, adenyl cyclase,and phosphodiesterase, and actuator proteins, e.g., protein kinase A andprotein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

For example, in one form of signal transduction, the effect of hormonebinding is activation of an enzyme, adenylate cyclase, inside the cell.Enzyme activation by hormones is dependent on the presence of thenucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors hasbeen characterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane α-helices connected byextracellular or cytoplasmic loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

G-protein coupled receptors have been characterized as including theseseven conserved hydrophobic stretches of about 20 to 30 amino acids,connecting at least eight divergent hydrophilic loops. The G-proteinfamily of coupled receptors includes dopamine receptors which bind toneuroleptic drugs used for treating psychotic and neurologicaldisorders. Other examples of members of this family include calcitonin,adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine,serotonin, histamine, thrombin, kinin, follicle stimulating hormone,opsins, endothelial differentiation gene-1 receptor and rhodopsins,odorant, cytomegalovirus receptors, etc.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (1989)).Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

Chemokines, also referred to as intercrine cytokines, are a subfamily ofstructurally and functionally related cytokines. These molecules are8-10 kd in size. In general, chemokines exhibit 20% to 75% homology atthe amino acid level and are characterized by four conserved cysteineresidues that form two disulfide bonds. Based on the arrangement of thefirst two cysteine residues, chemokines have been classified into twosubfamilies, alpha and beta. In the alpha subfamily, the first twocysteines are separated by one amino acid and hence are referred to asthe “C—X—C” subfamily. In the beta subfamily, the two cysteines are inan adjacent position and are, therefore, referred to as the “C—C”subfamily. Thus far, at least nine different members of this family havebeen identified in humans.

The intercrine cytokines exhibit a wide variety of functions. A hallmarkfeature is their ability to elicit chemotactic migration of distinctcell types, including monocytes, neutrophils, T lymphocytes, basophilsand fibroblasts. Many chemokines have proinflammatory activity and areinvolved in multiple steps during an inflammatory reaction. Theseactivities include stimulation of histamine release, lysosomal enzymeand leukotriene release, increased adherence of target immune cells toendothelial cells, enhanced binding of complement proteins, inducedexpression of granulocyte adhesion molecules and complement receptors,and respiratory burst. In addition to their involvement in inflammation,certain chemokines have been shown to exhibit other activities. Forexample, macrophage inflammatory protein 1 (MIP-1) is able to suppresshematopoietic stem cell proliferation, platelet factor-4 (PF-4) is apotent inhibitor of endothelial cell growth, Interleukin-8 (IL-8)promotes proliferation of keratinocytes, and GRO is an autocrine growthfactor for melanoma cells.

In light of the diverse biological activities, it is not surprising thatchemokines have been implicated in a number of physiological and diseaseconditions, including lymphocyte trafficking, wound healing,hematopoietic regulation and immunological disorders such as allergy,asthma and arthritis.

Thus, there is a need for polypeptides that modulate immune systemregulation, since disturbances of such regulation may be involved indiseases, disorders, and/or conditions relating to the immune system.Therefore, there is a need for identification and characterization ofsuch human polypeptides which can play a role in detecting, preventing,ameliorating or correcting such diseases, disorders, and/or conditions.

The G-protein Chemokine Receptor (CCR5) is a seven-pass transmembraneG-protein coupled receptor that is expressed in cells of the immunesystem such as, for example, macrophages, including immature dendriticcells such as Langerhans cells, and T cells, including Th0 and Th1effector cells. G-protein Chemokine Receptor (CCR5) has also beendetected in microglia, astrocytes, neurons, and vascular endothelialcells of the central nervous system (CNS). G-protein Chemokine Receptor(CCR5) is also expressed in monocyes and T cells in the synovial fluidof rheumatoid arthritis patients, and has also been implicated in otherforms of arthritis.

Ligands of G-protein Chemokine Receptor (CCR5) include MIP-1α, MIP-1β,MCP-1, MCP-2, MCP-3, MCP-4, RANTES, and Eotaxin. CCR5 is also a majorco-receptor for HIV, and may be also be recognized by other infectiousagents, such as other viruses, to allow entry into the cell. It wasrecently discovered that certain individuals harboring a mutation of theCCRS gene, were resistant to HIV infection despite multiple exposure tothe virus. This mutation abrogated expression of CCR5 at the cellsurface (Liu et al., Cell 86:1 (1996)).

HIV is currently the leading lethal infectious disease in the world,causing 2.6 million deaths in 1999. The number of deaths resulting fromHIV infection will continue to increase; In 1999, there were 5.6 millionnew cases of HIV infection and 33.6 million infected people living inthe world. Although there are currently 14 approved drugs to treat HIV,as many as one half of patents fail to be successfully (with successbeing defined as no detectable HIV RNA in serum (which in effect isequal to fewer than 50 copies/ml of HIV-1 RNA) treated after a one yeardrug regimen. The reasons for the inability of these drug regimens toeffectively treat HIV are several fold: use of certain drugs results inthe development of drug resistant HIV strains; some individuals areintolerant to certain drugs or the drugs have bad side effects; patientshave difficulty complying with complex dosing regimens; and the drugsmay not be able to access reservoirs of HIV in the body. Thus, thereremains a need in the art to develop improved HIV vaccines andtherapies.

SUMMARY OF THE INVENTION

The present invention relates to novel polynucleotides and the encodedpolypeptides of G-protein Chemokine Receptor (CCR5). Moreover, thepresent invention relates to vectors, host cells, antibodies, andrecombinant and synthetic methods for producing the polypeptides andpolynucleotides. Also provided are diagnostic methods for detectingdiseases, disorders, and/or conditions related to the polypeptides andpolynucleotides, and therapeutic methods for treating, preventing,and/or diagnosing such diseases, disorders, and/or conditions. Theinvention further relates to screening methods for identifying bindingpartners of G-protein Chemokine Receptor (CCR5).

In accordance with one aspect of the present invention, there areprovided novel mature receptor polypeptides as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof. The G-protein Chemokine Receptor (CCR5)polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding the G-proteinChemokine Receptor (CCR5) polypeptides of the present invention,including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogsthereof and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In accordance with a further aspect of the present invention, there areprovided processes for producing the G-protein Chemokine Receptor (CCR5)polypeptides by recombinant techniques comprising culturing recombinantprokaryotic and/or eukaryotic host cells, containing nucleic acidsequences encoding the receptor polypeptides of the present invention,under conditions promoting expression of said polypeptides andsubsequent recovery of said polypeptides.

In accordance with yet a further aspect of the present invention, thereare provided antibodies that bind the G-protein Chemokine Receptor(CCR5) polypeptides. The present invention encompasses antibodies(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) that immunospecifically bind toa G-protein Chemokine Receptor (CCR5) polypeptide or polypeptidefragment or variant of a G-protein Chemokine Receptor (CCR5). Inparticular, the invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that immunospecifically bind to a polypeptide orpolypeptide fragment or variant of human G-protein Chemokine Receptor(CCR5) such as those of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone.

The present invention relates to methods and compositions forpreventing, treating or ameliorating a disease or disorder comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that immunospecifically bind to a G-protein ChemokineReceptor (CCR5) or a fragment or variant thereof. In specificembodiments, the present invention relates to methods and compositionsfor preventing, treating or ameliorating a disease or disorderassociated with G-protein Chemokine Receptor (CCR5) function orG-protein Chemokine Receptor (CCR5) ligand function or aberrantG-protein Chemokine Receptor (CCR5) or G-protein Chemokine Receptor(CCR5) ligand expression, comprising administering to an animal,preferably a human, an effective amount of one or more antibodies orfragments or variants thereof, or related molecules, thatimmunospecifically bind to a G-protein Chemokine Receptor (CCR5) or afragment or variant thereof. In highly preferred embodiments, thepresent invention relates to antibody-based methods and compositions forpreventing, treating or ameliorating HIV infection and/or conditionsassociated with HIV infection. Other diseases and disorders which can betreated, prevented or ameliorated with the antibodies of the inventioninclude, but are not limited to, immune disorders (e.g., autoimmunedisorders such as multiple sclerosis, Grave's disease, and rheumatoidarthritis), neurodegenerative disorders (e.g., Alzheimer's disease)inflammatory disorders (e.g., asthma, allergic disorders, orinflammatory kidney diseases such as glomerulonephritis), infectiousdiseases (e.g., Hepatitis infections, herpes viral infections, and otherviral infections) and proliferative disorders.

The present invention also encompasses methods and compositions fordetecting, diagnosing, or prognosing diseases or disorders comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that immunospecifically bind to G-protein Chemokine Receptor(CCR5) or a fragment or variant thereof. In specific embodiments, thepresent invention also encompasses methods and compositions fordetecting, diagnosing, or prognosing diseases or disorders associatedwith G-protein Chemokine Receptor (CCR5) function or G-protein ChemokineReceptor (CCR5) ligand function or aberrant G-protein Chemokine Receptor(CCR5) or G-protein Chemokine Receptor (CCR5) ligand expression,comprising administering to an animal, preferably a human, an effectiveamount of one or more antibodies or fragments or variants thereof, orrelated molecules, that immunospecifically bind to G-protein ChemokineReceptor (CCR5) or a fragment or variant thereof. In highly preferredembodiments, the present invention relates to antibody-based methods andcompositions for detecting, diagnosing, or prognosing HIV infectionand/or conditions associated with HIV infection. Other diseases anddisorders which can be detected, diagnosed, or prognosed with theantibodies of the invention include, but are not limited to, immunedisorders (e.g., autoimmune disorders such as multiple sclerosis,Grave's disease, and rheumatoid arthritis), neurodegenerative disorders(e.g., Alzheimer's disease) inflammatory (e.g., asthma, allergicdisorders, or inflammatory kidney diseases such as glomerulonephritis),infectious diseases (e.g., Hepatitis infections, herpes viralinfections, and other viral infections) and proliferative disorders.

Another embodiment of the present invention includes the use of theantibodies of the invention as a diagnostic tool to monitor theexpression of G-protein Chemokine Receptor (CCR5) expression on cells.

The present invention also encompasses cell lines that expressantibodies that immunospecifically bind one or more G-protein ChemokineReceptor (CCR5) polypeptides (e.g., SEQ ID NO:2, or the polypeptideencoded by the deposited clone).

Further, the present invention encompasses the polynucleotides encodingthe antibodies expressed by such cell lines, as well as the amino acidsequences encoding the antibodies expressed by these cell lines.Molecules comprising, or alternatively consisting of, fragments orvariants of these antibodies (e.g., heavy chains, VH domains, VH CDRs,light chains, VL domains, or VL CDRs having an amino acid sequence ofany one of those expressed by an antibody-expressing cell line of theinvention, that immunospecifically bind to one or more G-proteinChemokine Receptor (CCR5) or fragments or variants thereof are alsoencompassed by the invention, as are nucleic acid molecules that encodethese antibodies and/or molecules. In highly preferred embodiments, thepresent invention encompasses antibodies, or fragments or variantsthereof, that bind to the extracellular regions/domains of one or moreG-protein Chemokine Receptor (CCR5) or fragments and variants thereof.

The present inventors have generated hybridoma cell lines that expressantibodies that immunospecifically bind one or more G-protein ChemokineReceptor (CCR5) polypeptides (e.g., SEQ ID NO:2 or the polypeptideencoded by the deposited clone). Thus, the invention encompasses thesecell lines, listed in Table 2 below which were deposited with theAmerican Type Culture Collection (“ATCC”) on the dates listed in Table 2and given the ATCC Deposit Numbers identified in Table 2 The ATCC islocated at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.The ATCC deposit was made pursuant to the terms of the Budapest Treatyon the international recognition of the deposit of microorganisms forpurposes of patent procedure.

Further, the present invention encompasses the polynucleotides encodingthe antibodies expressed by these cell lines, as well as the amino acidsequences encoding the antibodies expressed by these cell lines.Molecules comprising, or alternatively consisting of, fragments orvariants of these antibodies (e.g., heavy chains, VH domains, VH CDRs,light chains, VL domains, or VL CDRs having an amino acid sequence ofany one of those expressed by one or more cell lines referred to inTable 2), that immunospecifically bind to one or more G-proteinChemokine Receptor (CCR5) or fragments or variants thereof are alsoencompassed by the invention, as are nucleic acid molecules that encodethese antibodies and/or molecules. In highly preferred embodiments, thepresent invention encompasses antibodies, or fragments or variantsthereof, that bind to the extracellular regions/domains of one or moreG-protein Chemokine Receptor (CCR5) or fragments and variants thereof.

The present invention also provides antibodies that bind one or moreG-protein Chemokine Receptor (CCR5) polypeptides which are coupled to adetectable label, such as an enzyme, a fluorescent label, a luminescentlabel, or a bioluminescent label. The present invention also providesantibodies that bind one or more G-protein Chemokine Receptor (CCR5)polypeptides which are coupled to a therapeutic or cytotoxic agent. Thepresent invention also provides antibodies that bind one or moreG-protein Chemokine Receptor (CCR5) polypeptides which are coupled to aradioactive material.

The present invention further provides antibodies that inhibit orabolish the ability of HIV to bind to, enter into/fuse with (infect),and/or replicate in G-protein Chemokine Receptor (CCR5) expressingcells. In highly preferred embodiments of the present invention,anti-G-protein Chemokine Receptor (CCR5) antibodies of the presentinvention are used to treat, prevent or ameliorate HIV infection and/orconditions associated with HIV infection. In other highly preferredembodiments, anti-G-protein Chemokine Receptor (CCR5) antibodies of thepresent invention are administered to an individual alone or incombination with other therapeutic compounds, especially anti-retroviralagents, to treat, prevent or ameliorate HIV infection and/or conditionsassociated with HIV infection.

The present invention also provides antibodies that bind one or moreG-protein Chemokine Receptor (CCR5) polypeptides that act as eitherG-protein Chemokine Receptor (CCR5) agonists or G-protein ChemokineReceptor (CCR5) antagonists. In specific embodiments, the antibodies ofthe invention stimulate chemotaxis of G-protein Chemokine Receptor(CCR5) expressing cells. In other specific embodiments, the antibodiesof the invention inhibit G-protein Chemokine Receptor (CCR5) ligandbinding to a G-protein Chemokine Receptor (CCR5). In other specificembodiments, the antibodies of the invention upregulate G-proteinChemokine Receptor (CCR5) expression.

The present invention also provides antibodies that downregulateG-protein Chemokine Receptor (CCR5) expression. In still other specificembodiments, the anti-G-protein Chemokine Receptor (CCR5) antibodies ofthe invention downregulate G-protein Chemokine Receptor (CCR5)expression by promoting G-protein Chemokine Receptor (CCR5)internalization.

The present invention further provides antibodies that inhibit orabolish the binding of a G-protein Chemokine Receptor (CCR5) ligand,(e.g., MIP1-beta MIP-1alpha, MCP-1, MCP-2, MCP-3, MCP-4, RANTES, andEotaxin), to G-protein Chemokine Receptor (CCR5) expressing cells.

The present invention also provides for a nucleic acid molecule(s),generally isolated, encoding an antibody (including molecules, such asscFvs, VH domains, or VL domains, that comprise, or alternativelyconsist of, an antibody fragment or variant thereof) of the invention.The present invention also provides a host cell transformed with anucleic acid molecule encoding an antibody (including molecules, such asscFvs, VH domains, or VL domains, that comprise, or alternativelyconsist of, an antibody fragment or variant thereof) of the inventionand progeny thereof. The present invention also provides a method forthe production of an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof) ofthe invention. The present invention further provides a method ofexpressing an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof) ofthe invention from a nucleic acid molecule. These and other aspects ofthe invention are described in further detail below.

In another embodiment, the present invention provides vaccinescomprising, or alternatively consisting of, G-protein Chemokine Receptor(CCR5) polynucleotides or polypeptides or fragments, variants orderivatives thereof.

In accordance with another aspect of the present invention there areprovided methods of screening for compounds which bind to and activateor inhibit activation of the G-protein Chemokine Receptor (CCR5)polypeptides of the present invention.

In accordance with still another embodiment of the present inventionthere are provided processes of administering compounds to a host whichbind to and activate the receptor polypeptide of the present inventionwhich are useful in stimulating haematopoiesis, wound healing,coagulation, angiogenesis, to treat solid tumors, chronic infections,leukemia, T-cell mediated auto-immune diseases, parasitic infections,psoriasis, and to stimulate growth factor activity.

In accordance with another aspect of the present invention there isprovided a method of administering the receptor polypeptides of thepresent invention via gene therapy to treat conditions related tounderexpression of the polypeptides or underexpression of a ligand forthe G-protein Chemokine Receptor (CCR5) polypeptide.

In accordance with still another embodiment of the present inventionthere are provided processes of administering compounds to a host whichbind to and inhibit activation of the receptor polypeptides of thepresent invention which are useful in the prevention and/or treatment ofallergy, atherogenesis, anaphylaxis, malignancy, chronic and acuteinflammation, histamine and IgE-mediated allergic reactions,prostaglandin-independent fever, bone marrow failure, silicosis,sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilicsyndrome.

In accordance with yet another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to the polynucleotidesequences of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related tomutations in the nucleic acid sequences encoding such polypeptides andfor detecting an altered level of the soluble form of the receptorpolypeptides.

In accordance with yet a further aspect of the present invention, thereare provided processes for utilizing such receptor polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 shows the DNA sequence and the corresponding deduced amino acidsequence (SEQ ID NOs:1 and 2) of the G-protein coupled receptor of thepresent invention. The standard one-letter abbreviation for amino acidsis used. Sequencing was performed using a 373 Automated DNA sequencer(Applied Biosystems, Inc.).

FIG. 2 illustrates an amino acid alignment of the G-protein ChemokineReceptor (CCR5) of the present invention and the human MCP-1 receptor(SEQ ID NO:9). This figure shows the regions of identity between theamino acid sequence of the G-protein Chemokine Receptor (CCR5) proteinand the translation product of the human MCP-1 receptor A (MCP-1 RA)(SEQ ID NO:9), determined by BLAST analysis. Identical amino acidsbetween the two polypeptides are indicated by lines, while highlyconservative amino acid are indicated by colons and conservative aminoacids are indicated by periods. By examining the regions of identical,highly conserved and conserved amino acids, the skilled artisan canreadily identify conserved domains between the two polypeptides. Theseconserved domains are preferred embodiments of the present invention.

FIG. 3 shows an analysis of the G-protein Chemokine Receptor (CCR5)amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicityand hydrophobicity; amphipathic regions; flexible regions; antigenicindex and surface probability are shown, and all were generated usingthe default settings. In the “Antigenic Index or Jameson-Wolf” graph,the positive peaks indicate locations of the highly antigenic regions ofthe G-protein Chemokine Receptor (CCR5) protein, i.e., regions fromwhich epitope-bearing peptides of the invention can be obtained. Thedomains defined by these graphs are contemplated by the presentinvention.

The data presented in FIG. 3 are also represented in tabular form inTable 1. The columns are labeled with the headings “Res”, “Position”,and Roman Numerals I-XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 3, and Table 1:“Res”: amino acid residue of SEQ ID NO:2 and FIG. 1; “Position”:position of the corresponding residue within SEQ ID NO:2 and FIG. 1; I:Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III:Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn,Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

FIG. 4 shows the polynucleotide and amino acid sequence of the VH (SEQID NOs:59-60) and VL (SEQ ID NOs:61-62) domains of anti-CCR5 antibodyXF11.1D8. Each CDR is indicated by a line above the nucleotide sequence.See also, Table 6.

FIG. 5 shows the polynucleotide and amino acid sequence of the VH (SEQID NOs:63-64) and VL (SEQ ID NOs:65-66) domains of anti-CCR5 antibodyXF22.3C9 (i.e., XF22.3C9.6). Each CDR is indicated by a line above thenucleotide sequence. See also, Table 6.

FIG. 6 shows the polynucleotide and amino acid sequence of the VH (SEQID NOs:67-68) and VL (SEQ ID NOs:69-70) domains of anti-CCR5 antibodyXF22.9E6. Each CDR is indicated by a line above the nucleotide sequence.See also, Table 6.

DETAILED DESCRIPTION

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or for the mature polypeptide encoded by the clone deposited asATCC Deposit No. 97183 on Jun. 1, 1995. A sample of the deposited clone,which contains the open reading frame of the G-protein ChemokineReceptor (CCR5), has been obtained from the ATCC and has beenresequenced. The sequence data from the resequenced clone is shown inSEQ ID NO:21 and 22. SEQ ID NO:21 differs from SEQ ID NO: 1 at 5positions (nucleotides 320, 433, 442, 646, and 1289 of SEQ ID NO:1) SEQID NO:22 differs from SEQ ID NO:2 at 5 positions (amino acid residues21, 59, 62, 130, and 344).

The polynucleotide of this invention was discovered in a genomic libraryderived from human monocytes. It is structurally related to theG-protein-coupled receptor family. It contains an open reading frameencoding a protein of 352 amino acid residues. The protein exhibits thehighest degree of homology to a human MCP-1 receptor (SEQ ID NO:9) with70.1% identity and 82.9% similarity over a 347 amino acid stretch.

Polynucleotides of the invention include, but are not limited to, thenucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of theHDGNR10 deposited clone (ATCC Deposit Number 97183), the nucleotidesequence of SEQ ID NO:21), and/or fragments, variants or derivativesthereof.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ IDNO: 1) or the deposited clone.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orfor the mature polypeptide encoded by the deposited clone may include:only the coding sequence for the mature polypeptide; the coding sequencefor the mature polypeptide and additional coding sequence such as atransmembrane (TM) or intra-cellular domain; the coding sequence for themature polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequence 5′ and/or 3′of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or the polypeptide encoded by the deposited clone. The variant ofthe polynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIG. 1 (SEQ ID NO:2) or the polypeptideencoded by the deposited clone. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides may also encode for a soluble form of the G-proteinChemokine Receptor (CCR5) polypeptide which is the extracellular portionof the polypeptide which has been cleaved from the TM and intracellulardomain of the full-length polypeptide of the present invention.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promoter regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the DNAs of FIG. 1 (SEQ ID NO:1) orthe deposited clone.

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO: 1 or of the deposited clone, forexample, for recovery of the polynucleotide or as a diagnostic probe oras a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 or that encoded by the deposited clone as well asfragments thereof, which fragments have at least 30 bases and preferablyat least 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, or sell the deposited materials, and no such licenseis hereby granted.

The present invention further relates to a G-protein Chemokine Receptor(CCR5) polypeptide which has the deduced amino acid sequence of FIG. 1(SEQ ID NO:2) or which has the amino acid sequence encoded by thedeposited clone (SEQ ID NO:22), as well as fragments, analogs andderivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 or that encoded by the deposited clone, means apolypeptide which either retains substantially the same biologicalfunction or activity as such polypeptide, i.e. functions as a G-proteinChemokine Receptor (CCR5), or retains the ability to bind the ligand orthe receptor even though the polypeptide does not function as aG-protein Chemokine Receptor (CCR5), for example, a soluble form of thereceptor. An analog includes a proprotein which can be activated bycleavage of the proprotein portion to produce an active maturepolypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO: 2) or that encoded by the deposited clone may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptideor (v) one in which a fragment of the polypeptide is soluble, i.e. notmembrane bound, yet still binds ligands to the membrane bound receptor.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) or that encoded by thedeposited clone as well as polypeptides which have at least 70%similarity (preferably a 70% identity) to the polypeptide of SEQ IDNO:2) or to that encoded by the deposited clone and more preferably a90% similarity (more preferably a 90% identity) to the polypeptide ofSEQ ID NO:2) or to that encoded by the deposited clone and still morepreferably a 95% similarity (still more preferably a 90% identity) tothe polypeptide of SEQ ID NO:2 and to portions of such polypeptide withsuch portion of the polypeptide generally containing at least 30 aminoacids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis, therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2) or to that encoded by the deposited clone (in particular themature polypeptide) as well as polypeptides which have at least 70%similarity (preferably at least 70% identity) and more preferably atleast 90% similarity (more preferably at least 90% identity) and stillmore preferably at least 95% similarity (still more preferably at least95% identity) to the polypeptide of SEQ ID NO:2) or to that encoded bythe deposited clone and also include portions of such polypeptides withsuch portion of the polypeptide generally containing at least 30 aminoacids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli, lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenovirus; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., et al., Basic Methods inMolecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

The G-protein Chemokine Receptor (CCR5) polypeptides can be recoveredand purified from recombinant cell cultures by methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

The G-protein Chemokine Receptor (CCR5) of the present invention may beemployed in a process for screening for compounds which activate(agonists) or inhibit activation (antagonists) of the receptorpolypeptide of the present invention.

In general, such screening procedures involve providing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,drosophila or E. coli. In particular, a polynucleotide encoding thereceptor of the present invention is employed to transfect cells tothereby express the G-protein Chemokine Receptor (CCR5). The expressedreceptor is then contacted with a test compound to observe binding,stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which aretransfected to express the G-protein Chemokine Receptor (CCR5) of thepresent invention. Such a screening technique is described in PCT WO92/01810 published Feb. 6, 1992.

Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

The screen may be employed for determining a compound which activatesthe receptor by contacting such cells with compounds to be screened anddetermining whether such compound generates a signal, i.e., activatesthe receptor.

Other screening techniques include the use of cells which express theG-protein Chemokine Receptor (CCR5) (for example, transfected CHO cells)in a system which measures extracellular pH changes caused by receptoractivation, for example, as described in Science 246:181-296 (October1989). For example, compounds may be contacted with a cell whichexpresses the receptor polypeptide of the present invention and a secondmessenger response, e.g. signal transduction or pH changes, may bemeasured to determine whether the potential compound activates orinhibits the receptor.

Another such screening technique involves introducing RNA encoding theG-protein Chemokine Receptor (CCR5) into Xenopus oocytes to transientlyexpress the receptor. The receptor oocytes, may then be contacted withthe receptor ligand and a compound to be screened, followed by detectionof inhibition or activation of a calcium signal in the case of screeningfor compounds which are thought to inhibit activation of the receptor.

Another screening technique involves expressing the G-protein ChemokineReceptor (CCR5) in which the receptor is linked to a phospholipase C orD. As representative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the G-protein Chemokine Receptor(CCR5) such that the cell expresses the receptor on its surface andcontacting the cell with a compound in the presence of a labeled form ofa known ligand. The ligand can be labeled, e.g., by radioactivity. Theamount of labeled ligand bound to the receptors is measured, e.g., bymeasuring radioactivity of the receptors. If the compound binds to thereceptor as determined by a reduction of labeled ligand which binds tothe receptors, the binding of labeled ligand to the receptor isinhibited.

An antibody, or in some cases an oligopeptide, may activate a G-proteinChemokine Receptor (CCR5) of the present invention, by binding to theG-protein Chemokine Receptor (CCR5) and initiating second messengerresponse. Antibodies include anti-idiotypic antibodies which recognizeunique determinants generally associated with the antigen-binding siteof an antibody. Potential agonist compounds also include proteins whichare closely related to the ligand of the G-protein Chemokine Receptor(CCR5), e.g., a fragment of the ligand.

An antibody, or in some cases an oligopeptide, may antagonize aG-protein Chemokine Receptor (CCR5) of the present invention, by bindingto the G-protein Chemokine Receptor (CCR5) but failing to elicit asecond messenger response such that the activity of the G-proteinChemokine Receptor (CCR5) is prevented. Antibodies includeanti-idiotypic antibodies which recognize unique determinants generallyassociated with the antigen-binding site of an antibody. Potentialantagonist compounds also include proteins which are closely related tothe ligand of the G-protein Chemokine Receptor (CCR5), e.g., a fragmentof the ligand that has lost biological function and elicits no responsewhen binding to the G-protein Chemokine Receptor (CCR5).

An antisense construct prepared through the use of antisense technology,may be used to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix; see Lee et al., Nucl. AcidsRes. 6:3073 (1979); Cooney et al, Science 241:456 (1988); and Dervan etal., Science 251:1360 (1991)), thereby preventing transcription and theproduction of G-protein Chemokine Receptor (CCR5). The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofmRNA molecules into G-protein coupled receptor (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of G-protein Chemokine Receptor (CCR5).

A small molecule which binds to the G-protein Chemokine Receptor (CCR5),making it inaccessible to ligands such that normal biological activityis prevented, for example small peptides or peptide-like molecules, mayalso be used to inhibit activation of the receptor polypeptide of thepresent invention.

A soluble form of the G-protein Chemokine Receptor (CCR5), e.g. afragment of the receptors, may be used to inhibit activation of thereceptor by binding to the ligand to a polypeptide of the presentinvention and preventing the ligand from interacting with membrane boundG-protein Chemokine Receptor (CCR5).

The compounds which bind to and activate the G-protein ChemokineReceptor (CCR5) of the present invention may be employed to stimulatehaematopoiesis, wound healing, coagulation, angiogenesis, to treat solidtumors, chronic infections, leukemia, T-cell mediated auto-immunediseases, parasitic infections, psoriasis, and to stimulate growthfactor activity.

The compounds which bind to and inhibit the G-protein Chemokine Receptor(CCR5) of the present invention may be employed to treat allergy,atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation,histamine and IgE-mediated allergic reactions, prostaglandin-independentfever, bone marrow failure, silicosis, sarcoidosis, rheumatoidarthritis, shock and hyper-eosinophilic syndrome.

The compounds may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the compound and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thecompounds of the present invention may be employed in conjunction withother therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the pharmaceutical compositions will be administered in anamount of at least about 10 μg/kg body weight and in most cases theywill be administered in an amount not in excess of about 8 mg/Kg bodyweight per day. In most cases, the dosage is from about 10 μg/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The G-protein Chemokine Receptor (CCR5) polypeptides and antagonists oragonists which are polypeptides, may also be employed in accordance withthe present invention by expression of such polypeptides in vivo, whichis often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques 7:980-990 (1989), or any other promoter(e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and β-actinpromoters). Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, thymidine kinase (TK) promoters,and B19 parvovirus promoters. The selection of a suitable promoter willbe apparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe genes encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAM12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

The present invention also provides a method for determining whether aligand not known to be capable of binding to a G-protein ChemokineReceptor (CCR5) can bind to such receptor which comprises contacting amammalian cell which expresses a G-protein Chemokine Receptor (CCR5)with the ligand under conditions permitting binding of ligands to theG-protein Chemokine Receptor (CCR5), detecting the presence of a ligandwhich binds to the receptor and thereby determining whether the ligandbinds to the G-protein Chemokine Receptor (CCR5). The systemshereinabove described for determining agonists and/or antagonists mayalso be employed for determining ligands which bind to the receptor.

This invention also provides a method of detecting expression of aG-protein Chemokine Receptor (CCR5) polypeptide of the present inventionon the surface of a cell by detecting the presence of mRNA coding forthe receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with a nucleic acid probe comprising anucleic acid molecule of at least 10 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding the receptor under hybridizing conditions,detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the receptor by the cell.

The present invention also provides a method for identifying receptorsrelated to the receptor polypeptides of the present invention. Theserelated receptors may be identified by homology to a G-protein ChemokineReceptor (CCR5) polypeptide of the present invention, by low stringencycross hybridization, or by identifying receptors that interact withrelated natural or synthetic ligands and or elicit similar behaviorsafter genetic or pharmacological blockade of the chemokine receptorpolypeptides of the present invention.

Fragments of the genes may be used as a hybridization probe for a cDNAlibrary to isolate other genes which have a high sequence similarity tothe genes of the present invention, or which have similar biologicalactivity. Probes of this type are at least 20 bases, preferably at least30 bases and most preferably at least 50 bases or more. The probe mayalso be used to identify a cDNA clone corresponding to a full lengthtranscript and a genomic clone or clones that contain the complete geneof the present invention including regulatory and promoter regions,exons and introns. An example of a screen of this type comprisesisolating the coding region of the gene by using the known DNA sequenceto synthesize an oligonucleotide probe. Labeled oligonucleotides havinga sequence complementary to that of the genes of the present inventionare used to screen a library of human cDNA, genomic DNA or mRNA todetermine which members of the library the probe hybridizes to.

The present invention also contemplates the use of the genes of thepresent invention as a diagnostic, for example, some diseases resultfrom inherited defective genes. These genes can be detected by comparingthe sequences of the defective gene with that of a normal one.Subsequently, one can verify that a “mutant” gene is associated withabnormal receptor activity. In addition, one can insert mutant receptorgenes into a suitable vector for expression in a functional assay system(e.g., calorimetric assay, expression on MacConkey plates,complementation experiments, in a receptor deficient strain of HEK293cells) as yet another means to verify or identify mutations. Once“mutant” genes have been identified, one can then screen population forcarriers of the “mutant” receptor gene.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsused for diagnosis may be obtained from a patient's cells, including butnot limited to such as from blood, urine, saliva, tissue biopsy andautopsy material. The genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR (Saiki, et al., Nature324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used forthe same purpose. As an example, PCR primers complimentary to thenucleic acid of the instant invention can be used to identify andanalyze mutations in the gene of the present invention. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled RNA ofthe invention or alternatively, radiolabeled antisense DNA sequences ofthe invention. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures. Such a diagnostic would be particularly useful forprenatal or even neonatal testing.

Sequence differences between the reference gene and “mutants” may berevealed by the direct DNA sequencing method. In addition, cloned DNAsegments may be used as probes to detect specific DNA segments. Thesensitivity of this method is greatly enhanced when combined with PCR.For example, a sequence primer is used with double stranded PCR productor a single stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotide or by an automatic sequencing procedure withfluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alterations in the electrophoretic mobility of DNAfragments in gels with or without denaturing agents. Sequences changesat specific locations may also be revealed by nucleus protection assays,such RNase and S1 protection or the chemical cleavage method (e.g.Cotton, et al., PNAS, USA 85:4397-4401 (1985)).

In addition, some diseases are a result of, or are characterized bychanges in gene expression which can be detected by changes in the mRNA.Alternatively, the genes of the present invention can be used as areference to identify individuals expressing a decrease of functionsassociated with receptors of this type.

The present invention also relates to a diagnostic assay for detectingaltered levels of soluble forms of the G-protein Chemokine Receptor(CCR5) polypeptides of the present invention in various tissues. Assaysused to detect levels of the soluble receptor polypeptides in a samplederived from a host are well known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western blotanalysis and preferably as ELISA assay.

An ELISA assay initially comprises preparing an antibody specific toantigens of the G-protein Chemokine Receptor (CCR5) polypeptides,preferably a monoclonal antibody. In addition a reporter antibody isprepared against the monoclonal antibody. To the reporter antibody isattached a detectable reagent such as radioactivity, fluorescence or inthis example a horseradish peroxidase enzyme. A sample is now removedfrom a host and incubated on a solid support, e.g. a polystyrene dish,that binds the proteins in the sample. Any free protein binding sites onthe dish are then covered by incubating with a non-specific protein suchas bovine serum albumin. Next, the monoclonal antibody is incubated inthe dish during which time the monoclonal antibodies attach to anyG-protein Chemokine Receptor (CCR5) proteins attached to the polystyrenedish. All unbound monoclonal antibody is washed out with buffer. Thereporter antibody linked to horseradish peroxidase is now placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to G-protein Chemokine Receptor (CCR5) proteins.Unattached reporter antibody is then washed out. Peroxidase substratesare then added to the dish and the amount of color developed in a giventime period is a measurement of the amount of G-protein ChemokineReceptor (CCR5) proteins present in a given volume of patient samplewhen compared against a standard curve.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the clone. Computer analysis of the DNA ofthe deposited clone is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-DNAlibraries.

Fluorescence in situ hybridization (FISH) of a DNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with DNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature256:495-497 (1975)), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72 (1983)), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985), pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology 52:456-457 (1973).

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, a “secreted” or “soluble” G-protein ChemokineReceptor (CCR5) protein refers to a protein capable of being directed tothe ER, secretory vesicles, or the extracellular space as a result of asignal sequence, as well as a G-protein Chemokine Receptor (CCR5)protein released into the extracellular space without necessarilycontaining a signal sequence. If the G-protein Chemokine Receptor (CCR5)secreted protein is released into the extracellular space, the G-proteinChemokine Receptor (CCR5) secreted protein can undergo extracellularprocessing to produce a “mature” G-protein Chemokine Receptor (CCR5)protein. Release into the extracellular space can occur by manymechanisms, including exocytosis and proteolytic cleavage. Examples ofsecreted or soluble G-protein Chemokine Receptor (CCR5) protein includefragments comprising, or alternatively consisting of, portions of theG-protein Chemokine Receptor (CCR5) described herein. Preferred secretedor soluble fragments comprise an extracellular loop, an intracellularloop, the N-terminal extracellular domain, or the C-terminalintracellular domain, or fragments thereof. Additional preferredsecreted or soluble fragments comprise an epitope of the G-proteinChemokine Receptor (CCR5), such as described herein.

As used herein, a G-protein Chemokine Receptor (CCR5) “polynucleotide”refers to a molecule having a nucleic acid sequence contained in SEQ IDNO:1 or the G-protein Chemokine Receptor DNA contained within the clonedeposited with the ATCC. For example, the G-protein Chemokine Receptor(CCR5) polynucleotide can contain the nucleotide sequence of the fulllength genomic sequence, including the 5′ and 3′ untranslated sequences,the coding region, with or without the signal sequence, the secretedprotein coding region, as well as fragments, epitopes, domains, andvariants of the nucleic acid sequence. Moreover, as used herein, aG-protein Chemokine Receptor (CCR5) “polypeptide” refers to a moleculehaving the translated amino acid sequence generated from thepolynucleotide as broadly defined.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe G-protein Chemokine Receptor (CCR5) gene of interest in the genome).In other embodiments, the polynucleotides of the invention do notcontain the coding sequence of more than 1000, 500, 250, 100, 50, 25,20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

A representative clone containing the open reading frame of the sequencefor SEQ ID NO:1 was deposited with the American Type Culture Collection(“ATCC”) on Jun. 1, 1995, and was given the ATCC Deposit Number 97183.The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure.

A G-protein Chemokine Receptor (CCR5) “polynucleotide” also includesthose polynucleotides capable of hybridizing, under stringenthybridization conditions, to sequences contained in SEQ ID NO: 1, thecomplement thereof, or the DNA within the deposited clone. “Stringenthybridization conditions” refers to an overnight incubation at 42 degreeC. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65 degreeC.

Also contemplated are nucleic acid molecules that hybridize to theG-protein Chemokine Receptor (CCR5) polynucleotides under lowerstringency hybridization conditions. Changes in the stringency ofhybridization and signal detection are primarily accomplished throughthe manipulation of formamide concentration (lower percentages offormamide result in lowered stringency); salt conditions, ortemperature. For example, lower stringency conditions include anovernight incubation at 37 degree C. in a solution comprising 6×SSPE(20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30%formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lowerstringency, washes performed following stringent hybridization can bedone at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a DNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The G-protein Chemokine Receptor (CCR5) polynucleotide can be composedof any polyribonucleotide or polydeoxyribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example, G-proteinChemokine Receptor (CCR5) polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the G-protein Chemokine Receptor (CCR5) polynucleotides can becomposed of triple-stranded regions comprising RNA or DNA or both RNAand DNA. G-protein Chemokine Receptor (CCR5) polynucleotides may alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

G-protein Chemokine Receptor (CCR5) polypeptides can be composed ofamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres, and may contain amino acids other thanthe 20 gene-encoded amino acids. The G-protein Chemokine Receptor (CCR5)polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theG-protein Chemokine Receptor (CCR5) polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given G-proteinChemokine Receptor (CCR5) polypeptide. Also, a given G-protein ChemokineReceptor (CCR5) polypeptide may contain many types of modifications.G-protein Chemokine Receptor (CCR5) polypeptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic G-proteinChemokine Receptor (CCR5) polypeptides may result from posttranslationnatural processes or may be made by synthetic methods. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent isattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a G-protein Chemokine Receptor (CCR5)polynucleotide sequence while “SEQ ID NO:2” refers to a G-proteinChemokine Receptor (CCR5) polypeptide sequence.

A G-protein Chemokine Receptor (CCR5) polypeptide “having biologicalactivity” refers to polypeptides exhibiting activity similar, but notnecessarily identical to, an activity of a G-protein Chemokine Receptor(CCR5) polypeptide, including mature forms, as measured in a particularbiological assay, with or without dose dependency. In the case wheredose dependency does exist, it need not be identical to that of theG-protein Chemokine Receptor (CCR5) polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the G-protein Chemokine Receptor (CCR5) polypeptide (i.e.,the candidate polypeptide will exhibit greater activity or not more thanabout 25-fold less and, preferably, not more than about tenfold lessactivity, and most preferably, not more than about three-fold lessactivity relative to the G-protein Chemokine Receptor (CCR5)polypeptide.)

G-protein Chemokine Receptor (CCR5) Polynucleotides and Polypeptides

Clone HDGNR10 was isolated from a human monocyte genomic DNA library.This clone contains the entire coding region identified as SEQ ID NO:2.The deposited clone contains a DNA insert having a total of 1414nucleotides, which encodes a predicted open reading frame of 352 aminoacid residues. (See FIG. 1.) The open reading frame begins at aN-terminal methionine located at nucleotide position 259, and ends atthe last triplet coding for an amino acid at nucleotide position 1314.The stop codon is at positions 1315-1317.

Subsequent expression analysis also showed G-protein Chemokine Receptor(CCR5) expression in macrophages, including immature dendritic cellssuch as Langerhans cells, and T cells, including ThO and Th1 effectorcells, a pattern consistent with immune system-specific expression.G-protein Chemokine Receptor (CCR5) has also been detected in microglia,astrocytes, neurons, and vascular endothelial cells of the centralnervous system (CNS). G-protein Chemokine Receptor (CCR5) is alsoexpressed in monocyes and T cells in the synovial fluid of rheumatoidarthritis patients, and has also been implicated in other forms ofarthritis.

G-protein Coupled Chemokine Receptors. Using BLAST analysis, SEQ ID NO:2was found to be homologous to members of the G-Protein COUPLED ChemokineReceptor family. Particularly, SEQ ID NO:2 contains domains homologousto the translation product of the MonoMac 6 mRNA for human MCP-1receptor (MCP-1R) A (FIG. 2) (GenBank Accession No. U03882; SEQ IDNO:9), including the conserved transmembrane domain containing seventransmembrane segments characteristic of the G-protein coupled receptorfamily, which begins with amino acid 37 of SEQ ID NO:2 or thepolypeptide encoded by the deposited clone. G-protein Chemokine Receptor(CCR5) also includes the DRY motif, which is known to be required forsignal transduction, found in many G-protein coupled receptorsimmediately following the third transmembrane segment. Because MCP-1R isthought to be important in the immune system, the homology betweenMCP-1R and G-protein Chemokine Receptor (CCR5) suggests that G-proteinChemokine Receptor (CCR5) may also be involved in the immune system.

A second MCP-1R sequence has also been isolated which is identical tothe MCP-1RA sequence from the 5′ untranslated region through theputative seventh transmembrane domain but which contains a differentcytoplasmic tail. This second sequence, termed MCP-1RB, appears to be analternatively spliced version of MCP-1RA. It is described further inU.S. Pat. No. 5,707,815.

Domains. Using BLAST analysis, SEQ ID NO:2 was found to be homologous tomembers of the G-protein Chemokine Receptor (CCR5) family. Particularly,SEQ ID NO:2 contains domains homologous to the translation product ofthe MonoMac 6 mRNA for human MCP-1 receptor (MCP-1R) A (FIG. 2) (GenBankAccession No. U03882; SEQ ID NO:9), including the following conserveddomains: (a) a predicted N-terminal extracellular domain located atabout amino acids 1 to 36; (b) a predicted transmembrane domain locatedat about amino acids 37 to 305; and (c) a predicted C-terminalintracellular domain located at about amino acids 306 to 352. Thepredicted transmembrane domain includes: seven transmembrane segments atabout amino acids 37 to 58 (segment 1), 68 to 88 (segment 2), 103 to 124(segment 3), 142 to 166 (segment 4), 196 to 223 (segment 5), 236 to 260(segment 6), and 287 to 305 (segment 7); three intracellular loops atabout amino acids 59 to 67 (intracellular loop 1), 125 to 141(intracellular loop 2), and 224 to 235 (intracellular loop 3); and threeextracellular loops at about amino acids 89 to 102 (extracellular loop1), 167 to 195 (extracellular loop 2), and 261 to 274 (extracellularloop 3). These polypeptide fragments of G-protein Chemokine Receptor(CCR5) as defined above or as encoded by the deposited clone (SEQ IDNO:22) are specifically contemplated in the present invention, as arecombinations of these and other regions disclosed herein. Alsocontemplated are polypeptides which exclude one or more of thesedomains, segments, and loops. The “loops” are also referred to as“regions,” “domains,” and “portions” herein and in the art, e.g.,extracellular “regions”, intracellular “regions”, extracellular“domains”, and intracellular “domains”, extracellular “portions”, andintracellular “portions”.

SEQ ID NO:1 and the translated SEQ ID NO:2 are sufficiently accurate andotherwise suitable for a variety of uses well known in the art anddescribed further below. For instance, SEQ ID NO:1 is useful fordesigning nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO:1 or the DNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 may be used, for example, to generateantibodies which bind specifically to proteins G-protein ChemokineReceptor.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2, but also a sample of plasmid DNA containing a human DNA ofG-protein Chemokine Receptor (CCR5) deposited with the ATCC. Thenucleotide sequence of the deposited G-protein Chemokine Receptor (CCR5)clone can readily be determined by sequencing the deposited clone inaccordance with known methods. The predicted G-protein ChemokineReceptor (CCR5) amino acid sequence can then be verified from suchdeposits. Moreover, the amino acid sequence of the protein encoded bythe deposited clone can also be directly determined by peptidesequencing or by expressing the protein in a suitable host cellcontaining the deposited human G-protein Chemokine Receptor (CCR5) DNA,collecting the protein, and determining its sequence. A sample of thedeposited clone, which contains the open reading frame of the G-proteinChemokine Receptor (CCR5), has been obtained from the ATCC and has beenresequenced. The sequence data from the resequenced clone is shown inSEQ ID NO:21 and 22. SEQ ID NO:21 differs from SEQ ID NO:1 at 5positions (nucleotides 320, 433, 442, 646, and 1289 of SEQ ID NO:1) SEQID NO:22 differs from SEQ ID NO:2 at 5 positions (amino acid residues21, 59, 62, 130, and 344).

The present invention also relates to the G-protein Chemokine Receptor(CCR5) gene corresponding to SEQ ID NO:1, SEQ ID NO:2, or the depositedclone. The G-protein Chemokine Receptor (CCR5) gene can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include preparing probes or primers from thedisclosed sequence and identifying or amplifying the G-protein ChemokineReceptor (CCR5) gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:1, SEQ ID NO:2, or a the deposited clone,using information from the sequences disclosed herein or the clonesdeposited with the ATCC. For example, allelic variants and/or specieshomologs may be isolated and identified by making suitable probes orprimers from the sequences provided herein and screening a suitablenucleic acid source for allelic variants and/or the desired homologue.

The G-protein Chemokine Receptor (CCR5) polypeptides can be prepared inany suitable manner. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

The G-protein Chemokine Receptor (CCR5) polypeptides may be in the formof the secreted protein, including the mature form, or may be a part ofa larger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

G-protein Chemokine Receptor (CCR5) polypeptides are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a G-protein Chemokine Receptor (CCR5)polypeptide, including the secreted polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). G-protein Chemokine Receptor (CCR5)polypeptides also can be purified from natural, synthetic or recombinantsources using techniques described herein or otherwise known in the art,such as, for example, antibodies of the invention raised against theG-protein Chemokine Receptor (CCR5) protein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:1,and/or a clone contained in ATCC deposit 97183. The present inventionalso provides a polypeptide comprising, or alternatively, consisting of,the polypeptide sequence of SEQ ID NO:2 and/or a polypeptide encoded bythe clone contained in ATCC deposit 97183. Polynucleotides encoding apolypeptide comprising, or alternatively consisting of the polypeptidesequence of SEQ ID NO:2 and/or a polypeptide sequence encoded by theclone contained in ATCC deposit 97183 are also encompassed by theinvention.

Signal Sequences

As described herein, the present invention also encompasses fusions of asignal sequence with the polypeptide of SEQ ID NO:2, and fragmentsthereof, and/or the polypeptide encoded by the deposited clone, andfragments thereof, to direct secretion of the polypeptide or fragment.Polynucleotides encoding such fusions are also encompassed by theinvention.

The present invention also encompasses mature forms of the polypeptidehaving the sequence of SEQ ID NO:2, and fragments thereof, and/or thepolypeptide sequence encoded by the deposited clone, and fragmentsthereof. Polynucleotides encoding the mature forms (such as, forexample, the polynucleotide sequence in SEQ ID NO:1, and fragmentsthereof, and/or the polynucleotide sequence contained in the depositedclone, and fragments thereof) are also encompassed by the invention.

According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretary leader sequence which is cleaved from themature protein once export of the growing chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that cleavage specificity of a secretedprotein is ultimately determined by the primary structure of thecomplete protein, that is, it is inherent in the amino acid sequence ofthe polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

The deduced amino acid sequence of a secreted polypeptide can beanalyzed by a computer program called SignalP (Henrik Nielsen et al.,Protein Engineering 10:1-6 (1997)), which predicts the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Cleavage of a heterologous signal sequence in afusion protein may occur at the junction of the polypeptide sequences orcleavage may occur at a position on either side of the junction.Accordingly, the present invention provides secreted polypeptides havinga sequence shown in SEQ ID NO:2, and fragments thereof, which have anN-terminus beginning within 5 residues (i.e., + or −5 residues) of thepredicted cleavage point. Similarly, it is also recognized that in somecases, cleavage of the signal sequence from a secreted protein is notentirely uniform, resulting in more than one secreted species. Thesepolypeptides and fragments, and the polynucleotides encoding suchpolypeptides and fragments, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein or fragment produced by expression of the polynucleotidesequence of SEQ ID NO:1 or a fragment thereof and/or the polynucleotidesequence contained in the deposited clone or a fragment thereof, in amammalian cell (e.g., COS cells, as described below). Thesepolypeptides, and the polynucleotides encoding such polypeptides, arecontemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotidesequence disclosed in SEQ ID NO:1, the complementary strand thereto,and/or the sequence contained in a deposited clone.

The present invention also encompasses variants of the polypeptidesequence disclosed in SEQ ID NO:2 and/or encoded by a deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from theG-protein Chemokine Receptor (CCR5) polynucleotide or polypeptide, butretaining essential properties thereof. Generally, variants are overallclosely similar, and, in many regions, identical to the G-proteinChemokine Receptor (CCR5) polynucleotide or polypeptide.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:1 or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited clone or the complementary strand thereto, a nucleotidesequence encoding the polypeptide of SEQ ID NO:2, a nucleotide sequenceencoding the polypeptide encoded by the HDGNR10 deposited clone, and/orpolynucleotide fragments of any of these nucleic acid molecules (e.g.,those fragments described herein). Polynucleotides which hybridize tothese nucleic acid molecules under stringent hybridization conditions orlower stringency conditions are also encompassed by the invention, asare polypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:2, the polypeptide sequenceencoded by the deposited clone, and/or polypeptide fragments of any ofthese polypeptides (e.g., those fragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the G-protein ChemokineReceptor (CCR5) polypeptide. In other words, to obtain a nucleic acidhaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. The query sequencemay be an entire sequence shown of SEQ ID NO:1, the ORF (open readingframe) of the HDGNR10DNA in the deposited clone, or any fragmentspecified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences of SEQ ID NO:2 or to the amino acid sequenceencoded by the deposited clone can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).In a sequence alignment the query and subject sequences are either bothnucleotide sequences or both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The G-protein Chemokine Receptor (CCR5) variants may contain alterationsin the coding regions, non-coding regions, or both. Especially preferredare polynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.G-protein Chemokine Receptor (CCR5) polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

Naturally occurring G-protein Chemokine Receptor (CCR5) variants arecalled “allelic variants,” and refer to one of several alternate formsof a gene occupying a given locus on a chromosome of an organism. (GenesII, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelicvariants can vary at either the polynucleotide and/or polypeptide leveland are included in the present invention. Alternatively, non-naturallyoccurring variants may be produced by mutagenesis techniques or bydirect synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the G-protein Chemokine Receptor (CCR5) polypeptides.For instance, one or more amino acids can be deleted from the N-terminusor C-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993), reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes G-protein Chemokine Receptor (CCR5)polypeptide variants which show substantial biological activity. Suchvariants include deletions, insertions, inversions, repeats, andsubstitutions selected according to general rules known in the art so ashave little effect on activity.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, (e.g., encoding a polypeptide having the amino acidsequence of an N and/or C terminal deletion disclosed below as m-n ofSEQ ID NO:2) or which correspond to the polypeptide encoded by thedeposited clone, irrespective of whether they encode a polypeptidehaving G-protein Chemokine Receptor (CCR5) functional activity. This isbecause even where a particular nucleic acid molecule does not encode apolypeptide having G-protein Chemokine Receptor (CCR5) functionalactivity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving G-protein Chemokine Receptor (CCR5) functional activity include,inter alia, (1) isolating a G-protein Chemokine Receptor (CCR5) gene orallelic or splice variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the G-protein Chemokine Receptor (CCR5)gene, as described in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting G-protein Chemokine Receptor (CCR5) mRNAexpression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, which do, in fact, encode a polypeptide havingG-protein Chemokine Receptor (CCR5) functional activity. By “apolypeptide having G-protein Chemokine Receptor (CCR5) functionalactivity” is intended polypeptides exhibiting activity similar, but notnecessarily identical, to a functional activity of the G-proteinChemokine Receptor (CCR5) polypeptides of the present invention (e.g.,complete (full-length) G-protein Chemokine Receptor (CCR5), matureG-protein Chemokine Receptor (CCR5) and soluble G-protein ChemokineReceptor (CCR5) (e.g., having sequences contained in the extracellulardomain or regions of G-protein Chemokine Receptor) as measured, forexample, in a particular immunoassay or biological assay. For example, aG-protein Chemokine Receptor (CCR5) functional activity can routinely bemeasured by determining the ability of a G-protein Chemokine Receptor(CCR5) polypeptide to bind a G-protein Chemokine Receptor (CCR5) ligand.G-protein Chemokine Receptor (CCR5) functional activity may also bemeasured by determining the ability of a polypeptide, such as cognateligand which is free or expressed on a cell surface, to induce cellsexpressing the polypeptide.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the depositedclone, the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1), orfragments thereof, will encode polypeptides “having G-protein ChemokineReceptor (CCR5) functional activity.” In fact, since degenerate variantsof any of these nucleotide sequences all encode the same polypeptide, inmany instances, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving G-protein Chemokine Receptor (CCR5) functional activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly effectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

For example, site directed changes at the amino acid level of G-proteinChemokine Receptor (CCR5) can be made by replacing a particular aminoacid with a conservative amino acid. Preferred conservative mutations ofG-protein Chemokine Receptor (CCR5) (SEQ ID NO:2) include: M1 replacedwith A, G, I, L, S, T, or V; D2 replaced with E; Y3 replaced with F, orW; Q4 replaced with N; V5 replaced with A, G, I, L, S, T, or M; S6replaced with A, G, I, L, T, M, or V; S7 replaced with A, G, I, L, T, M,or V; I9 replaced with A, G, L, S, T, M, or V; Y10 replaced with F, orW; D11 replaced with E; I12 replaced with A, G, L, S, T, M, or V; N13replaced with Q; Y14 replaced with F, or W; Y15 replaced with F, or W;T16 replaced with A, G, I, L, S, M, or V; S17 replaced with A, G, I, L,T, M, or V; E18 replaced with D; K22 replaced with H, or R; I23 replacedwith A, G, L, S, T, M, or V; N24 replaced with Q; V25 replaced with A,G, I, L, S, T, or M; K26 replaced with H, or R; Q27 replaced with N; I28replaced with A, G, L, S, T, M, or V; A29 replaced with G, I, L, S, T,M, or V; A30 replaced with G, I, L, S, T, M, or V; R31 replaced with H,or K; L32 replaced with A, G, I, S, T, M, or V; L33 replaced with A, G,I, S, T, M, or V; L36 replaced with A, G, I, S, T, M, or V; Y37 replacedwith F, or W; S38 replaced with A, G, I, L, T, M, or V; L39 replacedwith A, G, I, S, T, M, or V; V40 replaced with A, G, I, L, S, T, or M;F41 replaced with W, or Y; I42 replaced with A, G, L, S, T, M, or V; F43replaced with W, or Y; G44 replaced with A, I, L, S, T, M, or V; F45replaced with W, or Y; V46 replaced with A, G, I, L, S, T, or M; G47replaced with A, I, L, S, T, M, or V; N48 replaced with Q; M49 replacedwith A, G, I, L, S, T, or V; L50 replaced with A, G, I, S, T, M, or V;V51 replaced with A, G, I, L, S, T, or M; I52 replaced with A, G, L, S,T, M, or V; L53 replaced with A, G, I, S, T, M, or V; I54 replaced withA, G, L, S, T, M, or V; L55 replaced with A, G, I, S, T, M, or V; 156replaced with A, G, L, S, T, M, or V; N57 replaced with Q; Q59 replacedwith N; R60 replaced with H, or K; L61 replaced with A, G, I, S, T, M,or V; E62 replaced with D; S63 replaced with A, G, I, L, T, M, or V; M64replaced with A, G, I, L, S, T, or V; T65 replaced with A, G, I, L, S,M, or V; D66 replaced with E; I67 replaced with A, G, L, S, T, M, or V;Y68 replaced with F, or W; L69 replaced with A, G, I, S, T, M, or V; L70replaced with A, G, I, S, T, M, or V; N71 replaced with Q; L72 replacedwith A, G, I, S, T, M, or V; A73 replaced with G, I, L, S, T, M, or V;I74 replaced with A, G, L, S, T, M, or V; S75 replaced with A, G, I, L,T, M, or V; D76 replaced with E; L77 replaced with A, G, I, S, T, M, orV; F78 replaced with W, or Y; F79 replaced with W, or Y; L80 replacedwith A, G, I, S, T, M, or V; L81 replaced with A, G, I, S, T, M, or V;T82 replaced with A, G, I, L, S, M, or V; V83 replaced with A, G, I, L,S, T, or M; F85 replaced with W, or Y; W86 replaced with F, or Y; A87replaced with G, I, L, S, T, M, or V; H88 replaced with K, or R; Y89replaced with F, or W; A90 replaced with G, I, L, S, T, M, or V; A91replaced with G, I, L, S, T, M, or V; A92 replaced with G, I, L, S, T,M, or V; Q93 replaced with N; W94 replaced with F, or Y; D95 replacedwith E; F96 replaced with W, or Y; G97 replaced with A, I, L, S, T, M,or V; N98 replaced with Q; T99 replaced with A, G, I, L, S, M, or V;M100 replaced with A, G, I, L, S, T, or V; Q102 replaced with N; L103replaced with A, G, I, S, T, M, or V; L104 replaced with A, G, I, S, T,M, or V; T105 replaced with A, G, I, L, S, M, or V; G106 replaced withA, I, L, S, T, M, or V; L107 replaced with A, G, I, S, T, M, or V; Y108replaced with F, or W; F109 replaced with W, or Y; I110 replaced with A,G, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; F112replaced with W, or Y; F113 replaced with W, or Y; S114 replaced with A,G, I, L, T, M, or V; G115 replaced with A, I, L, S, T, M, or V; I116replaced with A, G, L, S, T, M, or V; F117 replaced with W, or Y; F118replaced with W, or Y; I119 replaced with A, G, L, S, T, M, or V; I120replaced with A, G, L, S, T, M, or V; L121 replaced with A, G, I, S, T,M, or V; L122 replaced with A, G, I, S, T, M, or V; T123 replaced withA, G, I, L, S, M, or V; I124 replaced with A, G, L, S, T, M, or V; D125replaced with E; R126 replaced with H, or K; Y127 replaced with F, or W;L128 replaced with A, G, I, S, T, M, or V; A129 replaced with G, I, L,S, T, M, or V; I130 replaced with A, G, L, S, T, M, or V; V131 replacedwith A, G, I, L, S, T, or M; H132 replaced with K, or R; A133 replacedwith G, I, L, S, T, M, or V; V134 replaced with A, G, I, L, S, T, or M;F135 replaced with W, or Y; A136 replaced with G, I, L, S, T, M, or V;L137 replaced with A, G, I, S, T, M, or V; K138 replaced with H, or R;A139 replaced with G, I, L, S, T, M, or V; R140 replaced with H, or K;T141 replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I,L, S, T, or M; T143 replaced with A, G, I, L, S, M, or V; F144 replacedwith W, or Y; G145 replaced with A, I, L, S, T, M, or V; V146 replacedwith A, G, I, L, S, T, or M; V147 replaced with A, G, I, L, S, T, or M;T148 replaced with A, G, I, L, S, M, or V; S149 replaced with A, G, I,L, T, M, or V; V150 replaced with A, G, I, L, S, T, or M; I151 replacedwith A, G, L, S, T, M, or V; T152 replaced with A, G, I, L, S, M, or V;W153 replaced with F, or Y; V154 replaced with A, G, I, L, S, T, or M;V155 replaced with A, G, I, L, S, T, or M; A156 replaced with G, I, L,S, T, M, or V; V157 replaced with A, G, I, L, S, T, or M; F158 replacedwith W, or Y; A159 replaced with G, I, L, S, T, M, or V; S160 replacedwith A, G, I, L, T, M, or V; L161 replaced with A, G, I, S, T, M, or V;G163 replaced with A, I, L, S, T, M, or V; I164 replaced with A, G, L,S, T, M, or V; I165 replaced with A, G, L, S, T, M, or V; F166 replacedwith W, or Y; T167 replaced with A, G, I, L, S, M, or V; R168 replacedwith H, or K; S169 replaced with A, G, I, L, T, M, or V; Q170 replacedwith N; K171 replaced with H, or R; E172 replaced with D; G173 replacedwith A, I, L, S, T, M, or V; L174 replaced with A, G, I, S, T, M, or V;H175 replaced with K, or R; Y176 replaced with F, or W; T177 replacedwith A, G, I, L, S, M, or V; S179 replaced with A, G, I, L, T, M, or V;S180 replaced with A, G, I, L, T, M, or V; H181 replaced with K, or R;F182 replaced with W, or Y; Y184 replaced with F, or W; S185 replacedwith A, G, I, L, T, M, or V; Q186 replaced with N; Y187 replaced with F,or W; Q188 replaced with N; F189 replaced with W, or Y; W190 replacedwith F, or Y; K191 replaced with H, or R; N192 replaced with Q; F193replaced with W, or Y; Q194 replaced with N; T195 replaced with A, G, I,L, S, M, or V; L196 replaced with A, G, I, S, T, M, or V; K197 replacedwith H, or R; I198 replaced with A, G, L, S, T, M, or V; V199 replacedwith A, G, I, L, S, T, or M; I200 replaced with A, G, L, S, T, M, or V;L201 replaced with A, G, I, S, T, M, or V; G202 replaced with A, I, L,S, T, M, or V; L203 replaced with A, G, I, S, T, M, or V; V204 replacedwith A, G, I, L, S, T, or M; L205 replaced with A, G, I, S, T, M, or V;L207 replaced with A, G, I, S, T, M, or V; L208 replaced with A, G, I,S, T, M, or V; V209 replaced with A, G, I, L, S, T, or M; M210 replacedwith A, G, I, L, S, T, or V; V211 replaced with A, G, I, L, S, T, or M;I212 replaced with A, G, L, S, T, M, or V; Y214 replaced with F, or W;S215 replaced with A, G, I, L, T, M, or V; G216 replaced with A, I, L,S, T, M, or V; I217 replaced with A, G, L, S, T, M, or V; L218 replacedwith A, G, I, S, T, M, or V; K219 replaced with H, or R; T220 replacedwith A, G, I, L, S, M, or V; L221 replaced with A, G, I, S, T, M, or V;L222 replaced with A, G, I, S, T, M, or V; R223 replaced with H, or K;R225 replaced with H, or K; N226 replaced with Q; E227 replaced with D;K228 replaced with H, or R; K229 replaced with H, or R; R230 replacedwith H, or K; H231 replaced with K, or R; R232 replaced with H, or K;A233 replaced with G, I, L, S, T, M, or V; V234 replaced with A, G, I,L, S, T, or M; R235 replaced with H, or K; L236 replaced with A, G, I,S, T, M, or V; I237 replaced with A, G, L, S, T, M, or V; F238 replacedwith W, or Y; T239 replaced with A, G, I, L, S, M, or V; I240 replacedwith A, G, L, S, T, M, or V; M241 replaced with A, G, I, L, S, T, or V;I242 replaced with A, G, L, S, T, M, or V; V243 replaced with A, G, I,L, S, T, or M; Y244 replaced with F, or W; F245 replaced with W, or Y;L246 replaced with A, G, I, S, T, M, or V; F247 replaced with W, or Y;W248 replaced with F, or Y; A249 replaced with G, I, L, S, T, M, or V;Y251 replaced with F, or W; N252 replaced with Q; I253 replaced with A,G, L, S, T, M, or V; V254 replaced with A, G, I, L, S, T, or M; L255replaced with A, G, I, S, T, M, or V; L256 replaced with A, G, I, S, T,M, or V; L257 replaced with A, G, I, S, T, M, or V; N258 replaced withQ; T259 replaced with A, G, I, L, S, M, or V; F260 replaced with W, orY; Q261 replaced with N; E262 replaced with D; F263 replaced with W, orY; F264 replaced with W, or Y; G265 replaced with A, I, L, S, T, M, orV; L266 replaced with A, G, I, S, T, M, or V; N267 replaced with Q; N268replaced with Q; S270 replaced with A, G, I, L, T, M, or V; S271replaced with A, G, I, L, T, M, or V; S272 replaced with A, G, I, L, T,M, or V; N273 replaced with Q; R274 replaced with H, or K; L275 replacedwith A, G, I, S, T, M, or V; D276 replaced with E; Q277 replaced with N;A278 replaced with G, I, L, S, T, M, or V; M279 replaced with A, G, I,L, S, T, or V; Q280 replaced with N; V281 replaced with A, G, I, L, S,T, or M; T282 replaced with A, G, I, L, S, M, or V; E283 replaced withD; T284 replaced with A, G, I, L, S, M, or V; L285 replaced with A, G,I, S, T, M, or V; G286 replaced with A, I, L, S, T, M, or V; M287replaced with A, G, I, L, S, T, or V; T288 replaced with A, G, I, L, S,M, or V; H289 replaced with K, or R; I292 replaced with A, G, L, S, T,M, or V; N293 replaced with Q; I295 replaced with A, G, L, S, T, M, orV; I296 replaced with A, G, L, S, T, M, or V; Y297 replaced with F, orW; A298 replaced with G, I, L, S, T, M, or V; F299 replaced with W, orY; V300 replaced with A, G, I, L, S, T, or M; G301 replaced with A, I,L, S, T, M, or V; E302 replaced with D; K303 replaced with H, or R; F304replaced with W, or Y; R305 replaced with H, or K; N306 replaced with Q;Y307 replaced with F, or W; L308 replaced with A, G, 1, S, T, M, or V;L309 replaced with A, G, I, S, T, M, or V; V310 replaced with A, G, I,L, S, T, or M; F311 replaced with W, or Y; F312 replaced with W, or Y;Q313 replaced with N; K314 replaced with H, or R; H315 replaced with K,or R; I316 replaced with A, G, L, S, T, M, or V; A317 replaced with G,I, L, S, T, M, or V; K318 replaced with H, or R; R319 replaced with H,or K; F320 replaced with W, or Y; K322 replaced with H, or R; S325replaced with A, G, I, L, T, M, or V; I326 replaced with A, G, L, S, T,M, or V; F327 replaced with W, or Y; Q328 replaced with N; Q329 replacedwith N; E330 replaced with D; A331 replaced with G, I, L, S, T, M, or V;E333 replaced with D; R334 replaced with H, or K; A335 replaced with G,I, L, S, T, M, or V; S336 replaced with A, G, I, L, T, M, or V; S337replaced with A, G, I, L, T, M, or V; V338 replaced with A, G, I, L, S,T, or M; Y339 replaced with F, or W; T340 replaced with A, G, I, L, S,M, or V; R341 replaced with H, or K; S342 replaced with A, G, I, L, T,M, or V; T343 replaced with A, G, I, L, S, M, or V; G344 replaced withA, I, L, S, T, M, or V; E345 replaced with D; Q346 replaced with N; E347replaced with D; I348 replaced with A, G, L, S, T, M, or V; S349replaced with A, G, I, L, T, M, or V; V350 replaced with A, G, I, L, S,T, or M; G351 replaced with A, I, L, S, T, M, or V; and/or L352 replacedwith A, G, I, S, T, M, or V.

Preferred conservative mutations of G-protein Chemokine Receptor (CCR5)as encoded by the deposited HDGNR10 clone (SEQ ID NO:22) include: M1replaced with A, G, I, L, S, T, or V; D2 replaced with E; Y3 replacedwith F, or W; Q4 replaced with N; V5 replaced with A, G, I, L, S, T, orM; S6 replaced with A, G, I, L, T, M, or V; S7 replaced with A, G, I, L,T, M, or V; I9 replaced with A, G, L, S, T, M, or V; Y10 replaced withF, or W; D11 replaced with E; I12 replaced with A, G, L, S, T, M, or V;N13 replaced with Q; Y14 replaced with F, or W; Y15 replaced with F, orW; T16 replaced with A, G, I, L, S, M, or V; S17 replaced with A, G, I,L, T, M, or V; E18 replaced with D; Q21 replaced with N; K22 replacedwith H, or R; I23 replaced with A, G, L, S, T, M, or V; N24 replacedwith Q; V25 replaced with A, G, I, L, S, T, or M; K26 replaced with H,or R; Q27 replaced with N; I28 replaced with A, G, L, S, T, M, or V; A29replaced with G, I, L, S, T, M, or V; A30 replaced with G, I, L, S, T,M, or V; R31 replaced with H, or K; L32 replaced with A, G, I, S, T, M,or V; L33 replaced with A, G, I, S, T, M, or V; L36 replaced with A, G,I, S, T, M, or V; Y37 replaced with F, or W; S38 replaced with A, G, I,L, T, M, or V; L39 replaced with A, G, I, S, T, M, or V; V40 replacedwith A, G, I, L, S, T, or M; F41 replaced with W, or Y; I42 replacedwith A, G, L, S, T, M, or V; F43 replaced with W, or Y; G44 replacedwith A, I, L, S, T, M, or V; F45 replaced with W, or Y; V46 replacedwith A, G, I, L, S, T, or M; G47 replaced with A, I, L, S, T, M, or V;N48 replaced with Q; M49 replaced with A, G, I, L, S, T, or V; L50replaced with A, G, I, S, T, M, or V; V51 replaced with A, G, I, L, S,T, or M; I52 replaced with A, G, L, S, T, M, or V; L53 replaced with A,G, I, S, T, M, or V; I54 replaced with A, G, L, S, T, M, or V; L55replaced with A, G, I, S, T, M, or V; I56 replaced with A, G, L, S, T,M, or V; N57 replaced with Q; K59 replaced with H, or R; R60 replacedwith H, or K; L61 replaced with A, G, I, S, T, M, or V; K62 replacedwith H, or R; S63 replaced with A, G, I, L, T, M, or V; M64 replacedwith A, G, I, L, S, T, or V; T65 replaced with A, G, I, L, S, M, or V;D66 replaced with E; I67 replaced with A, G, L, S, T, M, or V; Y68replaced with F, or W; L69 replaced with A, G, I, S, T, M, or V; L70replaced with A, G, I, S, T, M, or V; N71 replaced with Q; L72 replacedwith A, G, I, S, T, M, or V; A73 replaced with G, I, L, S, T, M, or V;I74 replaced with A, G, L, S, T, M, or V; S75 replaced with A, G, I, L,T, M, or V; D76 replaced with E; L77 replaced with A, G, I, S, T, M, orV; F78 replaced with W, or Y; F79 replaced with W, or Y; L80 replacedwith A, G, I, S, T, M, or V; L81 replaced with A, G, I, S, T, M, or V;T82 replaced with A, G, I, L, S, M, or V; V83 replaced with A, G, I, L,S, T, or M; F85 replaced with W, or Y; W86 replaced with F, or Y; A87replaced with G, I, L, S, T, M, or V; H88 replaced with K, or R; Y89replaced with F, or W; A90 replaced with G, I, L, S, T, M, or V; A91replaced with G, I, L, S, T, M, or V; A92 replaced with G, I, L, S, T,M, or V; Q93 replaced with N; W94 replaced with F, or Y; D95 replacedwith E; F96 replaced with W, or Y; G97 replaced with A, I, L, S, T, M,or V; N98 replaced with Q; T99 replaced with A, G, I, L, S, M, or V;M100 replaced with A, G, I, L, S, T, or V; Q102 replaced with N; L103replaced with A, G, I, S, T, M, or V; L104 replaced with A, G, I, S, T,M, or V; T105 replaced with A, G, I, L, S, M, or V; G106 replaced withA, I, L, S, T, M, or V; L107 replaced with A, G, I, S, T, M, or V; Y108replaced with F, or W; F109 replaced with W, or Y; I110 replaced with A,G, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; F112replaced with W, or Y; F113 replaced with W, or Y; S114 replaced with A,G, I, L, T, M, or V; G115 replaced with A, I, L, S, T, M, or V; I116replaced with A, G, L, S, T, M, or V; F117 replaced with W, or Y; F118replaced with W, or Y; I119 replaced with A, G, L, S, T, M, or V; I120replaced with A, G, L, S, T, M, or V; L121 replaced with A, G, I, S, T,M, or V; L122 replaced with A, G, I, S, T, M, or V; T123 replaced withA, G, I, L, S, M, or V; I124 replaced with A, G, L, S, T, M, or V; D125replaced with E; R126 replaced with H, or K; Y127 replaced with F, or W;L128 replaced with A, G, I, S, T, M, or V; A129 replaced with G, I, L,S, T, M, or V; V130 replaced with A, G, I, L, S, T, or M; V131 replacedwith A, G, I, L, S, T, or M; H132 replaced with K, or R; A133 replacedwith G, I, L, S, T, M, or V; V134 replaced with A, G, I, L, S, T, or M;F135 replaced with W, or Y; A136 replaced with G, I, L, S, T, M, or V;L137 replaced with A, G, I, S, T, M, or V; K138 replaced with H, or R;A139 replaced with G, I, L, S, T, M, or V; R140 replaced with H, or K;T141 replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I,L, S, T, or M; T143 replaced with A, G, I, L, S, M, or V; F144 replacedwith W, or Y; G145 replaced with A, I, L, S, T, M, or V; V146 replacedwith A, G, I, L, S, T, or M; V147 replaced with A, G, I, L, S, T, or M;T148 replaced with A, G, I, L, S, M, or V; S149 replaced with A, G, I,L, T, M, or V; V150 replaced with A, G, I, L, S, T, or M; I151 replacedwith A, G, L, S, T, M, or V; T152 replaced with A, G, I, L, S, M, or V;W153 replaced with F, or Y; V154 replaced with A, G, I, L, S, T, or M;V155 replaced with A, G, I, L, S, T, or M; A156 replaced with G, I, L,S, T, M, or V; V157 replaced with A, G, I, L, S, T, or M; F158 replacedwith W, or Y; A159 replaced with G, I, L, S, T, M, or V; S160 replacedwith A, G, I, L, T, M, or V; L161 replaced with A, G, I, S, T, M, or V;G163 replaced with A, I, L, S, T, M, or V; I164 replaced with A, G, L,S, T, M, or V; I165 replaced with A, G, L, S, T, M, or V; F166 replacedwith W, or Y; T167 replaced with A, G, I, L, S, M, or V; R168 replacedwith H, or K; S169 replaced with A, G, I, L, T, M, or V; Q170 replacedwith N; K171 replaced with H, or R; E172 replaced with D; G173 replacedwith A, I, L, S, T, M, or V; L174 replaced with A, G, I, S, T, M, or V;H175 replaced with K, or R; Y176 replaced with F, or W; T177 replacedwith A, G, I, L, S, M, or V; S179 replaced with A, G, I, L, T, M, or V;S180 replaced with A, G, I, L, T, M, or V; H181 replaced with K, or R;F182 replaced with W, or Y; Y184 replaced with F, or W; S185 replacedwith A, G, I, L, T, M, or V; Q186 replaced with N; Y187 replaced with F,or W; Q188 replaced with N; F189 replaced with W, or Y; W190 replacedwith F, or Y; K191 replaced with H, or R; N192 replaced with Q; F193replaced with W, or Y; Q194 replaced with N; T195 replaced with A, G, I,L, S, M, or V; L196 replaced with A, G, I, S, T, M, or V; K197 replacedwith H, or R; I198 replaced with A, G, L, S, T, M, or V; V199 replacedwith A, G, I, L, S, T, or M; I200 replaced with A, G, L, S, T, M, or V;L201 replaced with A, G, I, S, T, M, or V; G202 replaced with A, I, L,S, T, M, or V; L203 replaced with A, G, I, S, T, M, or V; V204 replacedwith A, G, I, L, S, T, or M; L205 replaced with A, G, I, S, T, M, or V;L207 replaced with A, G, I, S, T, M, or V; L208 replaced with A, G, I,S, T, M, or V; V209 replaced with A, G, I, L, S, T, or M; M210 replacedwith A, G, I, L, S, T, or V; V211 replaced with A, G, I, L, S, T, or M;I212 replaced with A, G, L, S, T, M, or V; Y214 replaced with F, or W;S215 replaced with A, G, I, L, T, M, or V; G216 replaced with A, I, L,S, T, M, or V; I217 replaced with A, G, L, S, T, M, or V; L218 replacedwith A, G, I, S, T, M, or V; K219 replaced with H, or R; T220 replacedwith A, G, I, L, S, M, or V; L221 replaced with A, G, I, S, T, M, or V;L222 replaced with A, G, I, S, T, M, or V; R223 replaced with H, or K;R225 replaced with H, or K; N226 replaced with Q; E227 replaced with D;K228 replaced with H, or R; K229 replaced with H, or R; R230 replacedwith H, or K; H231 replaced with K, or R; R232 replaced with H, or K;A233 replaced with G, I, L, S, T, M, or V; V234 replaced with A, G, I,L, S, T, or M; R235 replaced with H, or K; L236 replaced with A, G, I,S, T, M, or V; I237 replaced with A, G, L, S, T, M, or V; F238 replacedwith W, or Y; T239 replaced with A, G, I, L, S, M, or V; I240 replacedwith A, G, L, S, T, M, or V; M241 replaced with A, G, I, L, S, T, or V;I242 replaced with A, G, L, S, T, M, or V; V243 replaced with A, G, I,L, S, T, or M; Y244 replaced with F, or W; F245 replaced with W, or Y;L246 replaced with A, G, I, S, T, M, or V; F247 replaced with W, or Y;W248 replaced with F, or Y; A249 replaced with G, I, L, S, T, M, or V;Y251 replaced with F, or W; N252 replaced with Q; I253 replaced with A,G, L, S, T, M, or V; V254 replaced with A, G, I, L, S, T, or M; L255replaced with A, G, I, S, T, M, or V; L256 replaced with A, G, I, S, T,M, or V; L257 replaced with A, G, I, S, T, M, or V; N258 replaced withQ; T259 replaced with A, G, I, L, S, M, or V; F260 replaced with W, orY; Q261 replaced with N; E262 replaced with D; F263 replaced with W, orY; F264 replaced with W, or Y; G265 replaced with A, I, L, S, T, M, orV; L266 replaced with A, G, I, S, T, M, or V; N267 replaced with Q; N268replaced with Q; S270 replaced with A, G, I, L, T, M, or V; S271replaced with A, G, I, L, T, M, or V; S272 replaced with A, G, I, L, T,M, or V; N273 replaced with Q; R274 replaced with H, or K; L275 replacedwith A, G, I, S, T, M, or V; D276 replaced with E; Q277 replaced with N;A278 replaced with G, I, L, S, T, M, or V; M279 replaced with A, G, I,L, S, T, or V; Q280 replaced with N; V281 replaced with A, G, I, L, S,T, or M; T282 replaced with A, G, I, L, S, M, or V; E283 replaced withD; T284 replaced with A, G, I, L, S, M, or V; L285 replaced with A, G,I, S, T, M, or V; G286 replaced with A, I, L, S, T, M, or V; M287replaced with A, G, I, L, S, T, or V; T288 replaced with A, G, I, L, S,M, or V; H289 replaced with K, or R; I292 replaced with A, G, L, S, T,M, or V; N293 replaced with Q; I295 replaced with A, G, L, S, T, M, orV; I296 replaced with A, G, L, S, T, M, or V; Y297 replaced with F, orW; A298 replaced with G, I, L, S, T, M, or V; F299 replaced with W, orY; V300 replaced with A, G, I, L, S, T, or M; G301 replaced with A, I,L, S, T, M, or V; E302 replaced with D; K303 replaced with H, or R; F304replaced with W, or Y; R305 replaced with H, or K; N306 replaced with Q;Y307 replaced with F, or W; L308 replaced with A, G, I, S, T, M, or V;L309 replaced with A, G, I, S, T, M, or V; V310 replaced with A, G, I,L, S, T, or M; F311 replaced with W, or Y; F312 replaced with W, or Y;Q313 replaced with N; K314 replaced with H, or R; H315 replaced with K,or R; I316 replaced with A, G, L, S, T, M, or V; A317 replaced with G,I, L, S, T, M, or V; K318 replaced with H, or R; R319 replaced with H,or K; F320 replaced with W, or Y; K322 replaced with H, or R; S325replaced with A, G, I, L, T, M, or V; I326 replaced with A, G, L, S, T,M, or V; F327 replaced with W, or Y; Q328 replaced with N; Q329 replacedwith N; E330 replaced with D; A331 replaced with G, I, L, S, T, M, or V;E333 replaced with D; R334 replaced with H, or K; A335 replaced with G,I, L, S, T, M, or V; S336 replaced with A, G, I, L, T, M, or V; S337replaced with A, G, I, L, T, M, or V; V338 replaced with A, G, I, L, S,T, or M; Y339 replaced with F, or W; T340 replaced with A, G, I, L, S,M, or V; R341 replaced with H, or K; S342 replaced with A, G, I, L, T,M, or V; T343 replaced with A, G, I, L, S, M, or V; E344 replaced withD; E345 replaced with D; Q346 replaced with N; E347 replaced with D;I348 replaced with A, G, L, S, T, M, or V; S349 replaced with A, G, I,L, T, M, or V; V350 replaced with A, G, I, L, S, T, or M; G351 replacedwith A, I, L, S, T, M, or V; and/or L352 replaced with A, G, I, S, T, M,or V.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or adecreased G-protein Chemokine Receptor (CCR5) activity or function,while the remaining G-protein Chemokine Receptor (CCR5) activities orfunctions are maintained. More preferably, the resulting constructs havemore than one increased and/or decreased G-protein Chemokine Receptor(CCR5) activity or function, while the remaining G-protein ChemokineReceptor (CCR5) activities or functions are maintained.

Besides conservative amino acid substitution, variants of G-proteinChemokine Receptor (CCR5) include (i) substitutions with one or more ofthe non-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, or leader or secretorysequence, or a sequence facilitating purification. Such variantpolypeptides are deemed to be within the scope of those skilled in theart from the teachings herein.

For example, G-protein Chemokine Receptor (CCR5) polypeptide variantscontaining amino acid substitutions of charged amino acids with othercharged or neutral amino acids may produce proteins with improvedcharacteristics, such as less aggregation. Aggregation of pharmaceuticalformulations both reduces activity and increases clearance due to theaggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol.2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Clelandet al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

For example, preferred non-conservative substitutions of G-proteinChemokine Receptor (CCR5) (SEQ ID NO:2) include: M1 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; Y3 replaced with D, E, H; K, R, N,Q, A, G, I, L, S, T, M, V, P, or C; Q4 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; V5 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S6 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S7 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P8replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; I9 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y10 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D11 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I12replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N13 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Y14 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y15 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T16 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S17 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E18 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; P19 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or C; C20 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P21 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K22 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I23 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; N24 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; V25 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K26 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; Q27 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; I28 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A30replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R31 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L32 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L33 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P34 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; P35 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; L36 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; Y37 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V40replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F41 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I42 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F43 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; G44 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F45 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; V46 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G47 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N48replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;M49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L50 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V51 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I52 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L55 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; I56 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; N57 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; C58 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or P; Q59 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; R60 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L61 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; E62 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; S63 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D66 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I67replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y68 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L69 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L70 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; N71 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A73 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I74replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S75 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; D76 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L77 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; F78 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; F79 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; L80 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L81 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T82replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V83 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P84 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; F85 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; W86 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A87 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; H88 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Y89 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; A90 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; A91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A92 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q93 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; W94replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D95replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;F96 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;G97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N98 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T99replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M100 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C100 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q102 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L103 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; L104 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; T105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; G106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L107replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y108 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F109 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I110 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F112 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; F113 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; S114 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G115 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I116replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F117 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F118 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I119 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I120 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L121 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L122 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T123replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I124 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; D125 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R126 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y127 replaced with D, E, H, K,R, N, Q, A, G, I, L, S, T, M, V, P, or C; L128 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; A129 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; I130 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V131 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H132 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A133 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V134 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F135 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; A136 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;K138 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A139 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R140 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V142 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T143 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; F144 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; G145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V146 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V147 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T148 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S149 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T152 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; W153 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V154 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V155 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; A156 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F158 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A159 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S160 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L161 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P162 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; G163 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; I164 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I165replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F166 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T167 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; R168 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; S169 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q170 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; K171 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; E172 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; G173 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L174 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;H175 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;Y176 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;T177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C178 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S179replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S180 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H181 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; F182 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; P183 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Y184 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S185 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Q186 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; Y187 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; Q188 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, F, W, Y, P, or C; F189 replaced with D, E, H, K, R, N,Q, A, G, I, L, S, T, M, V, P, or C; W190 replaced with D, E, H, K, R, N,Q, A, G, I, L, S, T, M, V, P, or C; K191 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; N192 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; F193 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; Q194 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; T195 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; L196 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; I198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I200 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L201 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; G202 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L203 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L205 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P206 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L207 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; L208 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; V209 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; M210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V211replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I212 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C213 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y214 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S215 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; G216 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; I217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L218 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K219 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T220 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L221 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L222 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R223 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; C224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; R225 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; N226 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; E227 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; K228 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; K229 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; R230 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; H231 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; R232 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; A233 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; V234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;R235 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I237 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F238 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T239 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I240 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V243 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y244 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F245 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L246 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F247 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; W248 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; A249 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P250 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; Y251 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; N252 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; I253 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V254 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L255replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L256 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L257 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; N258 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; T259 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; Q261 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; E262 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; F263 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; F264 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; G265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N267replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;N268 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; C269 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; S270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S271replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S272 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N273 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; R274 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L275 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; D276 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q277 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; A278 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; M279 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Q280 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; V281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T282replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E283 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T284 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L285 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; G286 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M287 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H289 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C290 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C291replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; I292 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N293replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;P294 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; I295 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I296replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y297 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A298 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F299 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; V300 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G301 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E302 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; K303 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; F304 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; R305 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; N306 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; Y307 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; L308 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L309 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V310 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F311 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F312 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q313 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K314 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H315 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I316 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; A317 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; K318 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; R319 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; F320 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; C321 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; K322 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; C323 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; C324 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; S325 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; I326 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; F327 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; Q328 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; Q329 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; E330 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; A331 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;P332 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; E333 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; R334 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A335 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S336replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S337 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V338 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Y339 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; T340 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; R341 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; S342 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T343replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G344 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; E345 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q346 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E347 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I348 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; S349 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; V350 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; G351 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L352replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.

Addition preferred non-conservative substitutions of G-protein ChemokineReceptor (CCR5) as encoded by the HDGNR10 deposited clone (SEQ ID NO:22)include: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;Y3 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;Q4 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; V5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S6 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S7 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; P8 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or C; I9 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; Y10 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; D11 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; I12 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; N13 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; Y14 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; Y15 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; T16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S17replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E18 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P19 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C20replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; Q21 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; K22 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; I23 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N24replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;V25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K26 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q27 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I28replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A29 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A30 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R31 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; L32 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L33 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P34replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; P35 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; L36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y37replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S38replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L39 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V40 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F41 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; I42 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F43 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; G44 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F45replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V46replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G47 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N48 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; M49 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; L50 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; V51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I52replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I54 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L55 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; I56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N57replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;C58 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; K59 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; R60 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K62replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S63replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M64 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T65 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D66 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; I67 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Y68 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; L69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L70replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N71 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L72 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A73 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I74 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S75 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; L77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F78 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F79 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L80 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L81 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T82 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;P84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; F85 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; W86 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; A87 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H88replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y89replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A90replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A91 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A92 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q93 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; W94 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; D95 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; F96 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; G97 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; N98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; T99 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; M100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C100replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; Q102 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; L103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L104replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T105 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G106 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L107 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y108 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; F109 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; I110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F112 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F113 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S114 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G115 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; I116 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F117 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; F118 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; I119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I120replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L121 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L122 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T123 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; I124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D125replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;R126 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;Y127 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;L128 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A129 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V130 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V131 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; H132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; A133 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F135 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A136 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L137 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; K138 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; A139 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R140 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T143 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F144 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G145 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V146 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S149 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V150 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I151 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;W153 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;V154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V155 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A156 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V157 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; F158 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; A159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L161 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P162 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G163 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; I164 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; I165 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; F166 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; T167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R168replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S169replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q170 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K171 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E172 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G173replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L174 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H175 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; Y176 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T177 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C178 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; S179 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S180 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;H181 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;F182 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;P183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; Y184 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; S185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q186replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;Y187 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;Q188 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; F189 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; W190 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; K191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N192 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; F193 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; Q194 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; T195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L196replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K197 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I198 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V199 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; I200 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G202replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L203 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V204 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L205 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; P206 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V209 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; M210 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V211 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; I212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;C213 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; Y214 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; S215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G216replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I217 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L218 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K219 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; T220 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L222replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R223 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C224 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R225replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N226replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E227 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; K228 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; K229 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; R230 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; H231 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; R232 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; A233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V234replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R235 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L236 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I237 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F238 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; T239 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; I240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M241replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I242 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V243 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Y244 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; F245 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; L246 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F247 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; W248 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; A249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P250replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; Y251 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; N252 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; I253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V254replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L255 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L256 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L257 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; N258 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; T259 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;Q261 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; E262 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; F263 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; F264 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; G265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L266replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N267 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N268 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C269replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; S270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S271replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S272 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N273 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; R274 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L275 replaced with D, E, H, K,R, N, Q, F; W, Y, P, or C; D276 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q277 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; A278 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; M279 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Q280 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; V281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T282replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E283 replaced withH, K, R, A, G, I, L, S; T, M, V, N, Q, F, W, Y, P, or C; T284 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L285 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; G286 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M287 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H289 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C290 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C291replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; I292 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N293replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;P294 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; I295 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I296replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y297 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A298 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F299 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; V300 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G301 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E302 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; K303 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; F304 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; R305 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; N306 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; Y307 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; L308 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L309 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V310 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F311 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F312 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q313 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K314 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H315 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I316 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; A317 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; K318 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; R319 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; F320 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; C321 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; K322 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; C323 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; C324 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; S325 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; I326 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; F327 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; Q328 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; Q329 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; E330 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; A331 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;P332 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; E333 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; R334 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A335 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S336replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S337 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V338 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Y339 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; T340 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; R341 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; S342 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T343replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E344 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E345 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q346replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E347 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I348 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S349replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V350 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G351 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; and/or L352 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or decreasedG-protein Chemokine Receptor (CCR5) activity or function, while theremaining G-protein Chemokine Receptor (CCR5) activities or functionsare maintained. More preferably, the resulting constructs have more thanone increased and/or decreased G-protein Chemokine Receptor (CCR5)activity or function, while the remaining G-protein Chemokine Receptor(CCR5) activities or functions are maintained.

Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and10) can be replaced with the substituted amino acids as described above(either conservative or nonconservative). The substituted amino acidscan occur in the full length, mature, or proprotein form of G-proteinChemokine Receptor (CCR5) protein, as well as the N- and C-terminaldeletion mutants, having the general formula m-n, listed below.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a G-protein Chemokine Receptor(CCR5) polypeptide having an amino acid sequence which contains at leastone amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a polypeptide to have an amino acid sequencewhich comprises the amino acid sequence of a G-protein ChemokineReceptor (CCR5) polypeptide, which contains at least one, but not morethan 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. Inspecific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of FIG. 1 or that encoded by thedeposited clone or fragments thereof (e.g., the mature form and/or otherfragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments ofthe polynucleotides of the invention. In the present invention, a“polynucleotide fragment” refers to a short polynucleotide having anucleic acid sequence which: is a portion of that contained in adeposited clone, or encoding the polypeptide encoded by deposited clone;is a portion of that shown in SEQ ID NO:1 or the complementary strandthereto, or is a portion of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2. The nucleotide fragments of the inventionare preferably at least about 15 nt, and more preferably at least about20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt, at least about 50 nt, at least about75 nt, or at least about 150 nt in length. A fragment “at least 20 nt inlength,” for example, is intended to include 20 or more contiguous basesfrom the HDGNR10DNA sequence contained in a deposited clone or thenucleotide sequence shown in SEQ ID NO:1. In this context “about”includes the particularly recited value, a value larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. These nucleotide fragments have uses that include, but are notlimited to, as diagnostic probes and primers as discussed herein. Ofcourse, larger fragments (e.g., 50, 150, 500, 600, 1000 nucleotides) arepreferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, or 1401 to the end of SEQ ID NO:1, orthe complementary strand thereto, or the HDGNR10DNA contained in thedeposited clone. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions are also encompassed by the invention, as are polypeptidesencoded by these polynucleotides. In the present invention, a“polypeptide fragment” refers to an amino acid sequence which is aportion of that contained in SEQ ID NO:2 or encoded by the HDGNR10DNAcontained in the deposited clone. Protein (polypeptide) fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80,81-100, 102-120, 121-140, 141-160, or 161 to the end of the codingregion. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. Inthis context “about” includes the particularly recited ranges or values,and ranges or values larger or smaller by several (5, 4, 3, 2, or 1)amino acids, at either extreme or at both extremes. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind G-protein ChemokineReceptor (CCR5) ligand) may still be retained. For example, the abilityof shortened G-protein Chemokine Receptor (CCR5) muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an G-proteinChemokine Receptor (CCR5) mutein with a large number of deletedN-terminal amino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six G-proteinChemokine Receptor (CCR5) amino acid residues may often evoke an immuneresponse.

Preferred polypeptide fragments include the secreted protein as well asthe mature form. Further preferred polypeptide fragments include thesecreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids,

Accordingly, polypeptide fragments include the secreted G-proteinChemokine Receptor (CCR5) protein as well as the mature form. Furtherpreferred polypeptide fragments include the secreted G-protein ChemokineReceptor (CCR5) protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted G-protein ChemokineReceptor (CCR5) polypeptide or the mature form. Similarly, any number ofamino acids, ranging from 1-30, can be deleted from the carboxy terminusof the secreted G-protein Chemokine Receptor (CCR5) protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

Particularly, N-terminal deletions of the G-protein Chemokine Receptor(CCR5) polypeptide can be described by the general formula m-352, wherem is an integer from 2 to 346, where m corresponds to the position ofthe amino acid residue identified in SEQ ID NO:2 or the polypeptideencoded by the deposited clone. More in particular, the inventionprovides polynucleotides encoding polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues ofN-terminal deletions of the polypeptide of the invention shown as SEQ IDNO:2 including polypeptides comprising the amino acid sequence ofresidues: D-2 to L-352; Y-3 to L-352; Q-4 to L-352; V-5 to L-352; S-6 toL-352; S-7 to L-352; P-8 to L-352; I-9 to L-352; Y-10 to L-352; D-11 toL-352; I-12 to L-352; N-13 to L-352; Y-14 to L-352; Y-15 to L-352; T-16to L-352; S-17 to L-352; E-18 to L-352; P-19 to L-352; C-20 to L-352;P-21 to L-352; K-22 to L-352; I-23 to L-352; N-24 to L-352; V-25 toL-352; K-26 to L-352; Q-27 to L-352; I-28 to L-352; A-29 to L-352; A-30to L-352; R-31 to L-352; L-32 to L-352; L-33, to L-352; P-34 to L-352;P-35 to L-352; L-36 to L-352; Y-37 to L-352; S-38 to L-352; L-39 toL-352; V40 to L-352; F-41 to L-352; I-42 to L-352; F-43 to L-352; G-44to L-352; F-45 to L-352; V-46 to L-352; G-47 to L-352; N-48 to L-352;M-49 to L-352; L-50 to L-352; V-51 to L-352; I-52 to L-352; L-53 toL-352; I-54 to L-352; L-55 to L-352; I-56 to L-352; N-57 to L-352; C-58to L-352; Q-59 to L-352; R-60 to L-352; L-61 to L-352; E-62 to L-352;S-63 to L-352; M-64 to L-352; T-65 to L-352; D-66 to L-352; I-67 toL-352; Y-68 to L-352; L-69 to L-352; L-70 to L-352; N-71 to L-352; L-72to L-352; A-73 to L-352; I-74 to L-352; S-75 to L-352; D-76 to L-352;L-77 to L-352; F-78 to L-352; F-79 to L-352; L-80 to L-352; L-81 toL-352; T-82 to L-352; V-83 to L-352; P-84 to L-352; F-85 to L-352; W-86to L-352; A-87 to L-352; H-88 to L-352; Y-89 to L-352; A-90 to L-352;A-91 to L-352; A-92 to L-352; Q-93 to L-352; W-94 to L-352; D-95 toL-352; F-96 to L-352; G-97 to L-352; N-98 to L-352; T-99 to L-352; M-100to L-352; C-101 to L-352; Q-102 to L-352; L-103 to L-352; L-104 toL-352; T-105 to L-352; G-106 to L-352; L-107 to L-352; Y-108 to L-352;F-109 to L-352; I-110 to L-352; G-111 to L-352; F-112 to L-352; F-113 toL-352; S-114 to L-352; G-115 to L-352; I-116 to L-352; F-117 to L-352;F-118 to L-352; I-119 to L-352; I-120 to L-352; L-121 to L-352; L-122 toL-352; T-123 to L-352; I-124 to L-352; D-125 to L-352; R-126 to L-352;Y-127 to L-352; L-128 to L-352; A-129 to L-352; I-130 to L-352; V-131 toL-352; H-132 to L-352; A-133 to L-352; V-134 to L-352; F-135 to L-352;A-136 to L-352; L-137 to L-352; K-138 to L-352; A-139 to L-352; R-140 toL-352; T-141 to L-352; V-142 to L-352; T-143 to L-352; F-144 to L-352;G-145 to L-352; V-146 to L-352; V-147 to L-352; T-148 to L-352; S-149 toL-352; V-150 to L-352; I-151 to L-352; T-152 to L-352; W-153 to L-352;V-154 to L-352; V-155 to L-352; A-156 to L-352; V-157 to L-352; F-158 toL-352; A-159 to L-352; S-160 to L-352; L-161 to L-352; P-162 to L-352;G-163 to L-352; I-164 to L-352; I-165 to L-352; F-166 to L-352; T-167 toL-352; R-168 to L-352; S-169 to L-352; Q-170 to L-352; K-171 to L-352;E-172 to L-352; G-173 to L-352; L-174 to L-352; H-175 to L-352; Y-176 toL-352; T-177 to L-352; C-178 to L-352; S-179 to L-352; S-180 to L-352;H-181 to L-352; F-182 to L-352; P-183 to L-352; Y-184 to L-352; S-185 toL-352; Q-186 to L-352; Y-187 to L-352; Q-188 to L-352; F-189 to L-352;W-190 to L-352; K-191 to L-352; N-192 to L-352; F-193 to L-352; Q-194 toL-352; T-195 to L-352; L-196 to L-352; K-197 to L-352; I-198 to L-352;V-199 to L-352; I-200 to L-352; L-201 to L-352; G-202 to L-352; L-203 toL-352; V-204 to L-352; L-205 to L-352; P-206 to L-352; L-207 to L-352;L-208 to L-352; V-209 to L-352; M-210 to L-352; V-211 to L-352; I-212 toL-352; C-213 to L-352; Y-214 to L-352; S-215 to L-352; G-216 to L-352;I-217 to L-352; L-218 to L-352; K-219 to L-352; T-220 to L-352; L-221 toL-352; L-222 to L-352; R-223 to L-352; C-224 to L-352; R-225 to L-352;N-226 to L-352; E-227 to L-352; K-228 to L-352; K-229 to L-352; R-230 toL-352; H-231 to L-352; R-232 to L-352; A-233 to L-352; V-234 to L-352;R-235 to L-352; L-236 to L-352; I-237 to L-352; F-238 to L-352; T-239 toL-352; I-240 to L-352; M-241 to L-352; I-242 to L-352; V-243 to L-352;Y-244 to L-352; F-245 to L-352; L-246 to L-352; F-247 to L-352; W-248 toL-352; A-249 to L-352; P-250 to L-352; Y-251 to L-352; N-252 to L-352;I-253 to L-352; V-254 to L-352; L-255 to L-352; L-256 to L-352; L-257 toL-352; N-258 to L-352; T-259 to L-352; F-260 to L-352; Q-261 to L-352;E-262 to L-352; F-263 to L-352; F-264 to L-352; G-265 to L-352; L-266 toL-352; N-267 to L-352; N-268 to L-352; C-269 to L-352; S-270 to L-352;S-271 to L-352; S-272 to L-352; N-273 to L-352; R-274 to L-352; L-275 toL-352; D-276 to L-352; Q-277 to L-352; A-278 to L-352; M-279 to L-352;Q-280 to L-352; V-281 to L-352; T-282 to L-352; E-283 to L-352; T-284 toL-352; L-285 to L-352; G-286 to L-352; M-287 to L-352; T-288 to L-352;H-289 to L-352; C-290 to L-352; C-291 to L-352; I-292 to L-352; N-293 toL-352; P-294 to L-352; I-295 to L-352; I-296 to L-352; Y-297 to L-352;A-298 to L-352; F-299 to L-352; V-300 to L-352; G-301 to L-352; E-302 toL-352; K-303 to L-352; F-304 to L-352; R-305 to L-352; N-306 to L-352;Y-307 to L-352; L-308 to L-352; L-309 to L-352; V-310 to L-352; F-311 toL-352; F-312 to L-352; Q-313 to L-352; K-314 to L-352; H-315 to L-352;I-316 to L-352; A-317 to L-352; K-318 to L-352; R-319 to L-352; F-320 toL-352; C-321 to L-352; K-322 to L-352; C-323 to L-352; C-324 to L-352;S-325 to L-352; I-326 to L-352; F-327 to L-352; Q-328 to L-352; Q-329 toL-352; E-330 to L-352; A-331 to L-352; P-332 to L-352; E-333 to L-352;R-334 to L-352; A-335 to L-352; S-336 to L-352; S-337 to L-352; V-338 toL-352; Y-339 to L-352; T-340 to L-352; R-341 to L-352; S-342 to L-352;T-343 to L-352; G-344 to L-352; E-345 to L-352; Q-346 to L-352; and/orE-347 to L-352 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

In addition, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of N-terminal deletions of the polypeptide of theinvention encoded by the deposited HDGNR10 clone (SEQ ID NO:22)including polypeptides comprising the amino acid sequence of residues:D-2 to L-352; Y-3 to L-352; Q4 to L-352; V-5 to L-352; S-6 to L-352; S-7to L-352; P-8 to L-352; I-9 to L-352; Y-10 to L-352; D-11 to L-352; I-12to L-352; N-13 to L-352; Y-14 to L-352; Y-15 to L-352; T-16 to L-352;S-17 to L-352; E-18 to L-352; P-19 to L-352; C-20 to L-352; Q-21 toL-352; K-22 to L-352; I-23 to L-352; N-24 to L-352; V-25 to L-352; K-26to L-352; Q-27 to L-352; 1-28 to L-352; A-29 to L-352; A-30 to L-352;R-31 to L-352; L-32 to L-352; L-33 to L-352; P-34 to L-352; P-35 toL-352; L-36 to L-352; Y-37 to L-352; S-38 to L-352; L-39 to L-352; V-40to L-352; F-41 to L-352; 142 to L-352; F-43 to L-352; G-44 to L-352; F45to L-352; V-46 to L-352; G47 to L-352; N-48 to L-352; M-49 to L-352;L-50 to L-352; V-51 to L-352; I-52 to L-352; L-53 to L-352; I-54 toL-352; L-55 to L-352; I-56 to L-352; N-57 to L-352; C-58 to L-352; K-59to L-352; R-60 to L-352; L-61 to L-352; K-62 to L-352; S-63 to L-352;M-64 to L-352; T-65 to L-352; D-66 to L-352; I-67 to L-352; Y-68 toL-352; L-69 to L-352; L-70 to L-352; N-71 to L-352; L-72 to L-352; A-73to L-352; I-74 to L-352; S-75 to L-352; D-76 to L-352; L-77 to L-352;F-78 to L-352; F-79 to L-352; L-80 to L-352; L-81 to L-352; T-82 toL-352; V-83 to L-352; P-84 to L-352; F-85 to L-352; W-86 to L-352; A-87to L-352; H-88 to L-352; Y-89 to L-352; A-90 to L-352; A-91 to L-352;A-92 to L-352; Q-93 to L-352; W-94 to L-352; D-95 to L-352; F-96 toL-352; G-97 to L-352; N-98 to L-352; T-99 to L-352; M-100 to L-352;C-101 to L-352; Q-102 to L-352; L-103 to L-352; L-104 to L-352; T-105 toL-352; G-106 to L-352; L-107 to L-352; Y-108 to L-352; F-109 to L-352;I-110 to L-352; G-ill to L-352; F-112 to L-352; F-113 to L-352; S-114 toL-352; G-115 to L-352; I-116 to L-352; F-117 to L-352; F-118 to L-352;I-119 to L-352; I-120 to L-352; L-121 to L-352; L-122 to L-352; T-123 toL-352; I-124 to L-352; D-125 to L-352; R-126 to L-352; Y-127 to L-352;L-128 to L-352; A-129 to L-352; V-130 to L-352; V-131 to L-352; H-132 toL-352; A-133 to L-352; V-134 to L-352; F-135 to L-352; A-136 to L-352;L-137 to L-352; K-138 to L-352; A-139 to L-352; R-140 to L-352; T-141 toL-352; V-142 to L-352; T-143 to L-352; F-144 to L-352; G-145 to L-352;V-146 to L-352; V-147 to L-352; T-148 to L-352; S-149 to L-352; V-150 toL-352; I-151 to L-352; T-152 to L-352; W-153 to L-352; V-154 to L-352;V-155 to L-352; A-156 to L-352; V-157 to L-352; F-158 to L-352; A-159 toL-352; S-160 to L-352; L-161 to L-352; P-162 to L-352; G-163 to L-352;I-164 to L-352; I-165 to L-352; F-166 to L-352; T-167 to L-352; R-168 toL-352; S-169 to L-352; Q-170 to L-352; K-171 to L-352; E-172 to L-352;G-173 to L-352; L-174 to L-352; H-175 to L-352; Y-176 to L-352; T-177 toL-352; C-178 to L-352; S-179 to L-352; S-180 to L-352; H-181 to L-352;F-182 to L-352; P-183 to L-352; Y-184 to L-352; S-185 to L-352; Q-186 toL-352; Y-187 to L-352; Q-188 to L-352; F-189 to L-352; W-190 to L-352;K-191 to L-352; N-192 to L-352; F-193 to L-352; Q-194 to L-352; T-195 toL-352; L-196 to L-352; K-197 to L-352; I-198 to L-352; V-199 to L-352;I-200 to L-352; L-201 to L-352; G-202 to L-352; L-203 to L-352; V-204 toL-352; L-205 to L-352; P-206 to L-352; L-207 to L-352; L-208 to L-352;V-209 to L-352; M-210 to L-352; V-211 to L-352; I-212 to L-352; C-213 toL-352; Y-214 to L-352; S-215 to L-352; G-216 to L-352; I-217 to L-352;L-218 to L-352; K-219 to L-352; T-220 to L-352; L-221 to L-352; L-222 toL-352; R-223 to L-352; C-224 to L-352; R-225 to L-352; N-226 to L-352;E-227 to L-352; K-228 to L-352; K-229 to L-352; R-230 to L-352; H1-231to L-352; R-232 to L-352; A-233 to L-352; V-234 to L-352; R-235 toL-352; L-236 to L-352; I-237 to L-352; F-238 to L-352; T-239 to L-352;I-240 to L-352; M-241 to L-352; I-242 to L-352; V-243 to L-352; Y-244 toL-352; F-245 to L-352; L-246 to L-352; F-247 to L-352; W-248 to L-352;A-249 to L-352; P-250 to L-352; Y-251 to L-352; N-252 to L-352; I-253 toL-352; V-254 to L-352; L-255 to L-352; L-256 to L-352; L-257 to L-352;N-258 to L-352; T-259 to L-352; F-260 to L-352; Q-261 to L-352; E-262 toL-352; F-263 to L-352; F-264 to L-352; G-265 to L-352; L-266 to L-352;N-267 to L-352; N-268 to L-352; C-269 to L-352; S-270 to L-352; S-271 toL-352; S-272 to L-352; N-273 to L-352; R-274 to L-352; L-275 to L-352;D-276 to L-352; Q-277 to L-352; A-278 to L-352; M-279 to L-352; Q-280 toL-352; V-281 to L-352; T-282 to L-352; E-283 to L-352; T-284 to L-352;L-285 to L-352; G-286 to L-352; M-287 to L-352; T-288 to L-352; H-289 toL-352; C-290 to L-352; C-291 to L-352; I-292 to L-352; N-293 to L-352;P-294 to L-352; I-295 to L-352; I-296 to L-352; Y-297 to L-352; A-298 toL-352; F-299 to L-352; V-300 to L-352; G-301 to L-352; E-302 to L-352;K-303 to L-352; F-304 to L-352; R-305 to L-352; N-306 to L-352; Y-307 toL-352; L-308 to L-352; L-309 to L-352; V-310 to L-352; F-311 to L-352;F-312 to L-352; Q-313 to L-352; K-314 to L-352; H-315 to L-352; I-316 toL-352; A-317 to L-352; K-318 to L-352; R-319 to L-352; F-320 to L-352;C-321 to L-352; K-322 to L-352; C-323 to L-352; C-324 to L-352; S-325 toL-352; I-326 to L-352; F-327 to L-352; Q-328 to L-352; Q-329 to L-352;E-330 to L-352; A-331 to L-352; P-332 to L-352; E-333 to L-352; R-334 toL-352; A-335 to L-352; S-336 to L-352; S-337 to L-352; V-338 to L-352;Y-339 to L-352; T-340 to L-352; R-341 to L-352; S-342 to L-352; T-343 toL-352; E-344 to L-352; E-345 to L-352; Q-346 to L-352; and/or E-347 toL-352 of SEQ ID NO:22. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the G-protein Chemokine Receptor (CCR5)polypeptide described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindG-protein Chemokine Receptor (CCR5) ligand) may still be retained. Forexample the ability of the shortened G-protein Chemokine Receptor (CCR5)mutein to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan G-protein Chemokine Receptor (CCR5) mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixG-protein Chemokine Receptor (CCR5) amino acid residues may often evokean immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the G-protein Chemokine Receptor (CCR5) polypeptide shown inFIG. 1 (SEQ ID NO:2) or of the polypeptide encoded by the depositedclone, as described by the general formula 1-n, where n is an integerfrom 6 to 346, where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2 or in the polypeptide encoded by the depositedclone. More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues of D-2 to G-351; D-2 to V-350; D-2 toS-349; D-2 to 1-348; D-2 to E-347; D-2 to Q-346; D-2 to E-345; D-2 toG-344; D-2 to T-343; D-2 to S-342; D-2 to R-341; D-2 to T-340; D-2 toY-339; D-2 to V-338; D-2 to S-337; D-2 to S-336; D-2 to A-335; D-2 toR-334; D-2 to E-333; D-2 to P-332; D-2 to A-331; D-2 to E-330; D-2 toQ-329; D-2 to Q-328; D-2 to F-327; D-2 to I-326; D-2 to S-325; D-2 toC-324; D-2 to C-323; D-2 to K-322; D-2 to C-321; D-2 to F-320; D-2 toR-319; D-2 to K-318; D-2 to A-317; D-2 to 1-316; D-2 to H-315; D-2 toK-314; D-2 to Q-313; D-2 to F-312; D-2 to F-311; D-2 to V-310; D-2 toL-309; D-2 to L-308; D-2 to Y-307; D-2 to N-306; D-2 to R-305; D-2 toF-304; D-2 to K-303; D-2 to E-302; D-2 to G-301; D-2 to V-300; D-2 toF-299; D-2 to A-298; D-2 to Y-297; D-2 to I-296; D-2 to I-295; D-2 toP-294; D-2 to N-293; D-2 to I-292; D-2 to C-291; D-2 to C-290; D-2 toH-289; D-2 to T-288; D-2 to M-287; D-2 to G-286; D-2 to L-285; D-2 toT-284; D-2 to E-283; D-2 to T-282; D-2 to V-281; D-2 to Q-280; D-2 toM-279; D-2 to A-278; D-2 to Q-277; D-2 to D-276; D-2 to L-275; D-2 toR-274; D-2 to N-273; D-2 to S-272; D-2 to S-271; D-2 to S-270; D-2 toC-269; D-2 to N-268; D-2 to N-267; D-2 to L-266; D-2 to G-265; D-2 toF-264; D-2 to F-263; D-2 to E-262; D-2 to Q-261; D-2 to F-260; D-2 toT-259; D-2 to N-258; D-2 to L-257; D-2 to L-256; D-2 to L-255; D-2 toV-254; D-2 to I-253; D-2 to N-252; D-2 to Y-251; D-2 to P-250; D-2 toA-249; D-2 to W-248; D-2 to F-247; D-2 to L-246; D-2 to F-245; D-2 toY-244; D-2 to V-243; D-2 to 1-242; D-2 to M-241; D-2 to I-240; D-2 toT-239; D-2 to F-238; D-2 to I-237; D-2 to L-236; D-2 to R-235; D-2 toV-234; D-2 to A-233; D-2 to R-232; D-2 to H-231; D-2 to R-230; D-2 toK-229; D-2 to K-228; D-2 to E-227; D-2 to N-226; D-2 to R-225; D-2 toC-224; D-2 to R-223; D-2 to L-222; D-2 to L-221; D-2 to T-220; D-2 toK-219; D-2 to L-218; D-2 to I-217; D-2 to G-216; D-2 to S-215; D-2 toY-214; D-2 to C-213; D-2 to I-212; D-2 to V-211; D-2 to M-210; D-2 toV-209; D-2 to L-208; D-2 to L-207; D-2 to P-206; D-2 to L-205; D-2 toV-204; D-2 to L-203; D-2 to G-202; D-2 to L-201; D-2 to I-200; D-2 toV-199; D-2 to I-198; D-2 to K-197; D-2 to L-196; D-2 to T-195; D-2 toQ-194; D-2 to F-193; D-2 to N-192; D-2 to K-191; D-2 to W-190; D-2 toF-189; D-2 to Q-188; D-2 to Y-187; D-2 to Q-186; D-2 to S-185; D-2 toY-184; D-2 to P-183; D-2 to F-182; D-2 to H-181; D-2 to S-180; D-2 toS-179; D-2 to C-178; D-2 to T-177; D-2 to Y-176; D-2 to H-175; D-2 toL-174; D-2 to G-173; D-2 to E-172; D-2 to K-171; D-2 to Q-170; D-2 toS-169; D-2 to R-168; D-2 to T-167; D-2 to F-166; D-2 to I-165; D-2 toI-164; D-2 to G-163; D-2 to P-162; D-2 to L-161; D-2 to S-160; D-2 toA-159; D-2 to F-158; D-2 to V-157; D-2 to A-156; D-2 to V-155; D-2 toV-154; D-2 to W-153; D-2 to T-152; D-2 to I-151; D-2 to V-150; D-2 toS-149; D-2 to T-148; D-2 to V-147; D-2 to V-146; D-2 to G-145; D-2 toF-144; D-2 to T-143; D-2 to V-142; D-2 to T-141; D-2 to R-140; D-2 toA-139; D-2 to K-138; D-2 to L-137; D-2 to A-136; D-2 to F-135; D-2 toV-134; D-2 to A-133; D-2 to H-132; D-2 to V-131; D-2 to I-130; D-2 toA-129; D-2 to L-128; D-2 to Y-127; D-2 to R-126; D-2 to D-125; D-2 toI-124; D-2 to T-123; D-2 to L-122; D-2 to L-121; D-2 to I-120; D-2 toI-119; D-2 to F-118; D-2 to F-117; D-2 to I-116; D-2 to G-115; D-2 toS-114; D-2 to F-113; D-2 to F-112; D-2 to G-111; D-2 to I-110; D-2 toF-109; D-2 to Y-108; D-2 to L-107; D-2 to G-106; D-2 to T-105; D-2 toL-104; D-2 to L-103; D-2 to Q-102; D-2 to C-101; D-2 to M-100; D-2 toT-99; D-2 to N-98; D-2 to G-97; D-2 to F-96; D-2 to D-95; D-2 to W-94;D-2 to Q-93; D-2 to A-92; D-2 to A-91; D-2 to A-90; D-2 to Y-89; D-2 toH-88; D-2 to A-87; D-2 to W-86; D-2 to F-85; D-2 to P-84; D-2 to V-83;D-2 to T-82; D-2 to L-81; D-2 to L-80; D-2 to F-79; D-2 to F-78; D-2 toL-77; D-2 to D-76; D-2 to S-75; D-2 to I-74; D-2 to A-73; D-2 to L-72;D-2 to N-71; D-2 to L-70; D-2 to L-69; D-2 to Y-68; D-2 to I-67; D-2 toD-66; D-2 to T-65; D-2 to M-64; D-2 to S-63; D-2 to E-62; D-2 to L-61;D-2 to R-60; D-2 to Q-59; D-2 to C-58; D-2 to N-57; D-2 to I-56; D-2 toL-55; D-2 to I-54; D-2 to L-53; D-2 to I-52; D-2 to V-51; D-2 to L-50;D-2 to M49; D-2 to N48; D-2 to G47; D-2 to V46; D-2 to F45; D-2 to G-44;D-2 to F-43; D-2 to 142; D-2 to F41; D-2 to V40; D-2 to L-39; D-2 toS-38; D-2 to Y-37; D-2 to L-36; D-2 to P-35; D-2 to P-34; D-2 to L-33;D-2 to L-32; D-2 to R-31; D-2 to A-30; D-2 to A-29; D-2 to I-28; D-2 toQ-27; D-2 to K-26; D-2 to V-25; D-2 to N-24; D-2 to I-23; D-2 to K-22;D-2 to P-21; D-2 to C-20; D-2 to P-19; D-2 to E-18; D-2 to S-17; D-2 toT-16; D-2 to Y-15; D-2 to Y-14; D-2 to N-13; D-2 to I-12; D-2 to D-11;D-2 to Y-10; D-2 to I-9; and/or D-2 to P-8 of SEQ ID NO:2. Moreover, amethionine may be added to the N-terminus of each of these C-terminalcontructs. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Additionally, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of D-2 to G-351; D-2 to V-350; D-2 to S-349; D-2 toI-348; D-2 to E-347; D-2 to Q-346; D-2 to E-345; D-2 to E-344; D-2 toT-343; D-2 to S-342; D-2 to R-341; D-2 to T-340; D-2 to Y-339; D-2 toV-338; D-2 to S-337; D-2 to S-336; D-2 to A-335; D-2 to R-334; D-2 toE-333; D-2 to P-332; D-2 to A-331; D-2 to E-330; D-2 to Q-329; D-2 toQ-328; D-2 to F-327; D-2 to I-326; D-2 to S-325; D-2 to C-324; D-2 toC-323; D-2 to K-322; D-2 to C-321; D-2 to F-320; D-2 to R-319; D-2 toK-318; D-2 to A-317; D-2 to I-316; D-2 to H-315; D-2 to K-314; D-2 toQ-313; D-2 to F-312; D-2 to F-311; D-2 to V-310; D-2 to L-309; D-2 toL-308; D-2 to Y-307; D-2 to N-306; D-2 to R-305; D-2 to F-304; D-2 toK-303; D-2 to E-302; D-2 to G-301; D-2 to V-300; D-2 to F-299; D-2 toA-298; D-2 to Y-297; D-2 to I-296; D-2 to I-295; D-2 to P-294; D-2 toN-293; D-2 to I-292; D-2 to C-291; D-2 to C-290; D-2 to H-289; D-2 toT-288; D-2 to M-287; D-2 to G-286; D-2 to L-285; D-2 to T-284; D-2 toE-283; D-2 to T-282; D-2 to V-281; D-2 to Q-280; D-2 to M-279; D-2 toA-278; D-2 to Q-277; D-2 to D-276; D-2 to L-275; D-2 to R-274; D-2 toN-273; D-2 to S-272; D-2 to S-271; D-2 to S-270; D-2 to C-269; D-2 toN-268; D-2 to N-267; D-2 to L-266; D-2 to G-265; D-2 to F-264; D-2 toF-263; D-2 to E-262; D-2 to Q-261; D-2 to F-260; D-2 to T-259; D-2 toN-258; D-2 to L-257; D-2 to L-256; D-2 to L-255; D-2 to V-254; D-2 toI-253; D-2 to N-252; D-2 to Y-251; D-2 to P-250; D-2 to A-249; D-2 toW-248; D-2 to F-247; D-2 to L-246; D-2 to F-245; D-2 to Y-244; D-2 toV-243; D-2 to I-242; D-2 to M-241; D-2 to I-240; D-2 to T-239; D-2 toF-238; D-2 to I-237; D-2 to L-236; D-2 to R-235; D-2 to V-234; D-2 toA-233; D-2 to R-232; D-2 to H-231; D-2 to R-230; D-2 to K-229; D-2 toK-228; D-2 to E-227; D-2 to N-226; D-2 to R-225; D-2 to C-224; D-2 toR-223; D-2 to L-222; D-2 to L-221; D-2 to T-220; D-2 to K-219; D-2 toL-218; D-2 to I-217; D-2 to G-216; D-2 to S-215; D-2 to Y-214; D-2 toC-213; D-2 to I-212; D-2 to V-211; D-2 to M-210; D-2 to V-209; D-2 toL-208; D-2 to L-207; D-2 to P-206; D-2 to L-205; D-2 to V-204; D-2 toL-203; D-2 to G-202; D-2 to L-201; D-2 to I-200; D-2 to V-199; D-2 toI-198; D-2 to K-197; D-2 to L-196; D-2 to T-195; D-2 to Q-194; D-2 toF-193; D-2 to N-192; D-2 to K-191; D-2 to W-190; D-2 to F-189; D-2 toQ-188; D-2 to Y-187; D-2 to Q-186; D-2 to S-185; D-2 to Y-184; D-2 toP-183; D-2 to F-182; D-2 to H-181; D-2 to S-180; D-2 to S-179; D-2 toC-178; D-2 to T-177; D-2 to Y-176; D-2 to H-175; D-2 to L-174; D-2 toG-173; D-2 to E-172; D-2 to K-171; D-2 to Q-170; D-2 to S-169; D-2 toR-168; D-2 to T-167; D-2 to F-166; D-2 to I-165; D-2 to I-164; D-2 toG-163; D-2 to P-162; D-2 to L-161; D-2 to S-160; D-2 to A-159; D-2 toF-158; D-2 to V-157; D-2 to A-156; D-2 to V-155; D-2 to V-154; D-2 toW-153; D-2 to T-152; D-2 to I-151; D-2 to V-150; D-2 to S-149; D-2 toT-148; D-2 to V-147; D-2 to V-146; D-2 to G-145; D-2 to F-144; D-2 toT-143; D-2 to V-142; D-2 to T-141; D-2 to R-140; D-2 to A-139; D-2 toK-138; D-2 to L-137; D-2 to A-136; D-2 to F-135; D-2 to V-134; D-2 toA-133; D-2 to H-132; D-2 to V-131; D-2 to V-130; D-2 to A-129; D-2 toL-128; D-2 to Y-127; D-2 to R-126; D-2 to D-125; D-2 to I-124; D-2 toT-123; D-2 to L-122; D-2 to L-121; D-2 to I-120; D-2 to I-119; D-2 toF-118; D-2 to F-117; D-2 to I-116; D-2 to G-115; D-2 to S-114; D-2 toF-113; D-2 to F-112; D-2 to G-111; D-2 to I-110; D-2 to F-109; D-2 toY-108; D-2 to L-107; D-2 to G-106; D-2 to T-105; D-2 to L-104; D-2 toL-103; D-2 to Q-102; D-2 to C-101; D-2 to M-100; D-2 to T-99; D-2 toN-98; D-2 to G-97; D-2 to F-96; D-2 to D-95; D-2 to W-94; D-2 to Q-93;D-2 to A-92; D-2 to A-91; D-2 to A-90; D-2 to Y-89; D-2 to H-88; D-2 toA-87; D-2 to W-86; D-2 to F-85; D-2 to P-84; D-2 to V-83; D-2 to T-82;D-2 to L-81; D-2 to L-80; D-2 to F-79; D-2 to F-78; D-2 to L-77; D-2 toD-76; D-2 to S-75; D-2 to I-74; D-2 to A-73; D-2 to L-72; D-2 to N-71;D-2 to L-70; D-2 to L-69; D-2 to Y-68; D-2 to I-67; D-2 to D-66; D-2 toT-65; D-2 to M-64; D-2 to S-63; D-2 to K-62; D-2 to L-61; D-2 to R-60;D-2 to K-59; D-2 to C-58; D-2 to N-57; D-2 to I-56; D-2 to L-55; D-2 toI-54; D-2 to L-53; D-2 to I-52; D-2 to V-51; D-2 to L-50; D-2 to M-49;D-2 to N-48; D-2 to G-47; D-2 to V-46; D-2 to F45; D-2 to G-44; D-2 toF-43; D-2 to I-42; D-2 to F-41; D-2 to V-40; D-2 to L-39; D-2 to S-38;D-2 to Y-37; D-2 to L-36; D-2 to P-35; D-2 to P-34; D-2 to L-33; D-2 toL-32; D-2 to R-31; D-2 to A-30; D-2 to A-29; D-2 to I-28; D-2 to Q-27;D-2 to K-26; D-2 to V-25; D7-2 to N-24; D-2 to I-23; D-2 to K-22; D-2 toQ-21; D-2 to C-20; D-2 to P-19; D-2 to E-18; D-2 to S-17; D-2 to T-16;D-2 to Y-15; D-2 to Y-14; D-2 to N-13; D-2 to I-12; D-2 to D-11; D-2 toY-10; D-2 to I-9; and/or D-2 to P-8 of SEQ ID NO:22. Moreover, amethionine may be added to the N-terminus of each of these C-terminalcontructs. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the G-protein Chemokine Receptor (CCR5)polypeptide described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence.

In addition, any of the above listed N- or C-terminal deletions can becombined to produce a N- and C-terminal deleted G-protein ChemokineReceptor (CCR5) polypeptide. The invention also provides polypeptideshaving one or more amino acids deleted from both the amino and thecarboxyl termini, which may be described generally as having residuesm-n of SEQ ID NO:2 or of the polypeptide encoded by the deposited clone,where n and m are integers as described above. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete G-protein Chemokine Receptor(CCR5) amino acid sequence encoded by the clone contained in ATCCDeposit No. 97183, where this portion excludes any integer of amino acidresidues from 1 to about 342 amino acids from the amino terminus of thecomplete amino acid sequence encoded by the clone contained in ATCCDeposit No. 97183, or any integer of amino acid residues from 1 to about342 amino acids from the carboxy terminus, or any combination of theabove amino terminal and carboxy terminal deletions, of the completeamino acid sequence encoded by the clone contained in ATCC Deposit No.97183. Polynucleotides encoding all of the above deletion mutantpolypeptide forms also are provided.

The present application is also directed to proteins containingpolypeptides at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the G-protein Chemokine Receptor (CCR5) polypeptidesequence set forth herein m-n. In preferred embodiments, the applicationis directed to proteins containing polypeptides at least 90%, 95%, 96%,97%, 98% or 99% identical to polypeptides having the amino acid sequenceof the specific G-protein Chemokine Receptor (CCR5) N- and C-terminaldeletions recited herein. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

Additional preferred polypeptide fragments comprise, or alternativelyconsist of, the amino acid sequence of residues: M-1 to Y-15; D-2 toT-16; Y-3 to S-17; Q-4 to E-18; V-5 to P-19; S-6 to C-20; S-7 to P-21;P-8 to K-22; I-9 to I-23; Y-10 to N-24; D-11 to V-25; I-12 to K-26; N-13to Q-27; Y-14 to I-28; Y-15 to A-29; T-16 to A-30; S-17 to R-31; E-18 toL-32; P-19 to L-33; C-20 to P-34; P-21 to P-35; K-22 to L-36; I-23 toY-37; N-24 to S-38; V-25 to L-39; K-26 to V-40; Q-27 to F-41; I-28 toI-42; A-29 to F-43; A-30 to G-44; R-31 to F-45; L-32 to V-46; L-33 toG-47; P-34 to N-48; P-35 to M-49; L-36 to L-50; Y-37 to V-51; S-38 toI-52; L-39 to L-53; V-40 to I-54; F-41 to L-55; I-42 to I-56; F43 toN-57; G44 to C-58; F45 to Q-59; V-46 to R-60; G47 to L-61; N48 to E-62;M-49 to S-63; L-50 to M-64; V-51 to T-65; I-52 to D-66; L-53 to I-67;I-54 to Y-68; L-55 to L-69; I-56 to L-70; N-57 to N-71; C-58 to L-72;Q-59 to A-73; R-60 to I-74; L-61 to S-75; E-62 to D-76; S-63 to L-77;M-64 to F-78; T-65 to F-79; D-66 to L-80; I-67 to L-81; Y-68 to T-82;L-69 to V-83; L-70 to P-84; N-71 to F-85; L-72 to W-86; A-73 to A-87;I-74 to H-88; S-75 to Y-89; D-76 to A-90; L-77 to A-91; F-78 to A-92;F-79 to Q-93; L-80 to W-94; L-81 to D-95; T-82 to F-96; V-83 to G-97;P-84 to N-98; F-85 to T-99; W-86 to M-100; A-87 to C-101; H-88 to Q-102;Y-89 to L-103; A-90 to L-104; A-91 to T-105; A-92 to G-106; Q-93 toL-107; W-94 to Y-108; D-95 to F-109; F-96 to I-110; G-97 to G-111; N-98to F-112; T-99 to F-113; M-100 to S-114; C-101 to G-115; Q-102 to I-116;L-103 to F-117; L-104 to F-118; T-105 to I-119; G-106 to I-120; L-107 toL-121; Y-108 to L-122; F-109 to T-123; I-110 to I-124; G-111 to D-125;F-112 to R-126; F-113 to Y-127; S-114 to L-128; G-115 to A-129; I-116 toI-130; F-117 to V-131; F-118 to H-132; I-119 to A-133; I-120 to V-134;L-121 to F-135; L-122 to A-136; T-123 to L-137; I-124 to K-138; D-125 toA-139; R-126 to R-140; Y-127 to T-141; L-128 to V-142; A-129 to T-143;I-130 to F-144; V-131 to G-145; H-132 to V-146; A-133 to V-147; V-134 toT-148; F-135 to S-149; A-136 to V-150; L-137 to I-151; K-138 to T-152;A-139 to W-153; R-140 to V-154; T-141 to V-155; V-142 to A-156; T-143 toV-157; F-144 to F-158; G-145 to A-159; V-146 to S-160; V-147 to L-161;T-148 to P-162; S-149 to G-163; V-150 to I-164; I-151 to I-165; T-152 toF-166; W-153 to T-167; V-154 to R-168; V-155 to S-169; A-156 to Q-170;V-157 to K-171; F-158 to E-172; A-159 to G-173; S-160 to L-174; L-161 toH-175; P-162 to Y-176; G-163 to T-177; I-164 to C-178; I-165 to S-179;F-166 to S-180; T-167 to H-181; R-168 to F-182; S-169 to P-183; Q-170 toY-184; K-171 to S-185; E-172 to Q-186; G-173 to Y-187; L-174 to Q-188;H-175 to F-189; Y-176 to W-190; T-177 to K-191; C-178 to N-192; S-179 toF-193; S-180 to Q-194; H-181 to T-195; F-182 to L-196; P-183 to K-197;Y-184 to I-198; S-185 to V-199; Q-186 to I-200; Y-187 to L-201; Q-188 toG-202; F-189 to L-203; W-190 to V-204; K-191 to L-205; N-192 to P-206;F-193 to L-207; Q-194 to L-208; T-195 to V-209; L-196 to M-210; K-197 toV-211; I-198 to I-212; V-199 to C-213; I-200 to Y-214; L-201 to S-215;G-202 to G-216; L-203 to I-217; V-204 to L-218; L-205 to K-219; P-206 toT-220; L-207 to L-221; L-208 to L-222; V-209 to R-223; M-210 to C-224;V-211 to R-225; I-212 to N-226; C-213 to E-227; Y-214 to K-228; S-215 toK-229; G-216 to R-230; I-217 to H-231; L-218 to R-232; K-219 to A-233;T-220 to V-234; L-221 to R-235; L-222 to L-236; R-223 to I-237; C-224 toF-238; R-225 to T-239; N-226 to I-240; E-227 to M-241; K-228 to I-242;K-229 to V-243; R-230 to Y-244; H-231 to F-245; R-232 to L-246; A-233 toF-247; V-234 to W-248; R-235 to A-249; L-236 to P-250; I-237 to Y-251;F-238 to N-252; T-239 to I-253; I-240 to V-254; M-241 to L-255; I-242 toL-256; V-243 to L-257; Y-244 to N-258; F-245 to T-259; L-246 to F-260;F-247 to Q-261; W-248 to E-262; A-249 to F-263; P-250 to F-264; Y-251 toG-265; N-252 to L-266; I-253 to N-267; V-254 to N-268; L-255 to C-269;L-256 to S-270; L-257 to S-271; N-258 to S-272; T-259 to N-273; F-260 toR-274; Q-261 to L-275; E-262 to D-276; F-263 to Q-277; F-264 to A-278;G-265 to M-279; L-266 to Q-280; N-267 to V-281; N-268 to T-282; C-269 toE-283; S-270 to T-284; S-271 to L-285; S-272 to G-286; N-273 to M-287;R-274 to T-288; L-275 to H-289; D-276 to C-290; Q-277 to C-291; A-278 toI-292; M-279 to N-293; Q-280 to P-294; V-281 to I-295; T-282 to I-296;E-283 to Y-297; T-284 to A-298; L-285 to F-299; G-286 to V-300; M-287 toG-301; T-288 to E-302; H-289 to K-303; C-290 to F-304; C-291 to R-305;I-292 to N-306; N-293 to Y-307; P-294 to L-308; I-295 to L-309; I-296 toV-310; Y-297 to F-311; A-298 to F-312; F-299 to Q-313; V-300 to K-314;G-301 to H-315; E-302 to I-316; K-303 to A-317; F-304 to K-318; R-305 toR-319; N-306 to F-320; Y-307 to C-321; L-308 to K-322; L-309 to C-323;V-310 to C-324; F-311 to S-325; F-312 to I-326; Q-313 to F-327; K-314 toQ-328; H-315 to Q-329; I-316 to E-330; A-317 to A-331; K-318 to P-332;R-319 to E-333; F-320 to R-334; C-321 to A-335; K-322 to S-336; C-323 toS-337; C-324 to V-338; S-325 to Y-339; I-326 to T-340; F-327 to R-341;Q-328 to S-342; Q-329 to T-343; E-330 to G-344; A-331 to E-345; P-332 toQ-346; E-333 to E-347; R-334 to I-348; A-335 to S-349; S-336 to V-350;S-337 to G-351; and/or V-338 to L-352 of SEQ ID NO:2.

Additional preferred polypeptide fragments comprise, or alternativelyconsist of, the amino acid sequence of residues: M-1 to Y-15; D-2 toT-16; Y-3 to S-17; Q4 to E-18; V-5 to P-19; S-6 to C-20; S-7 to Q-21;P-8 to K-22; I-9 to I-23; Y-10 to N-24; D-11 to V-25; I-12 to K-26; N-13to Q-27; Y-14 to I-28; Y-15 to A-29; T-16 to A-30; S-17 to R-31; E-18 toL-32; P-19 to L-33; C-20 to P-34; Q-21 to P-35; K-22 to L-36; I-23 toY-37; N-24 to S-38; V-25 to L-39; K-26 to V-40; Q-27 to F41; I-28 to142; A-29 to F43; A-30 to G-44; R-31 to F-45; L-32 to V46; L-33 to G47;P-34 to N-48; P-35 to M-49; L-36 to L-50; Y-37 to V-51; S-38 to I-52;L-39 to L-53; V40 to I-54; F-41 to L-55; I-42 to I-56; F43 to N-57; G44to C-58; F45 to K-59; V-46 to R-60; G-47 to L-61; N-48 to K-62; M49 toS-63; L-50 to M-64; V-51 to T-65; I-52 to D-66; L-53 to I-67; I-54 toY-68; L-55 to L-69; I-56 to L-70; N-57 to N-71; C-58 to L-72; K-59 toA-73; R-60 to I-74; L-61 to S-75; K-62 to D-76; S-63 to L-77; M-64 toF-78; T-65 to F-79; D-66 to L-80; I-67 to L-81; Y-68 to T-82; L-69 toV-83; L-70 to P-84; N-71 to F-85; L-72 to W-86; A-73 to A-87; I-74 toH-88; S-75 to Y-89; D-76 to A-90; L-77 to A-91; F-78 to A-92; F-79 toQ-93; L-80 to W-94; L-81 to D-95; T-82 to F-96; V-83 to G-97; P-84 toN-98; F-85 to T-99; W-86 to M-100; A-87 to C-101; H-88 to Q-102; Y-89 toL-103; A-90 to L-104; A-91 to T-105; A-92 to G-106; Q-93 to L-107; W-94to Y-108; D-95 to F-109; F-96 to I-110; G-97 to G-111; N-98 to F-112;T-99 to F-113; M-100 to S-114; C-101 to G-115; Q-102 to I-116; L-103 toF-117; L-104 to F-118; T-105 to I-119; G-106 to I-120; L-107 to L-121;Y-108 to L-122; F-109 to T-123; I-110 to I-124; G-111 to D-125; F-112 toR-126; F-113 to Y-127; S-114 to L-128; G-115 to A-129; I-116 to V-130;F-117 to V-131; F-118 to H-132; I-119 to A-133; I-120 to V-134; L-121 toF-135; L-122 to A-136; T-123 to L-137; I-124 to K-138; D-125 to A-139;R-126 to R-140; Y-127 to T-141; L-128 to V-142; A-129 to T-143; V-130 toF-144; V-131 to G-145; H-132 to V-146; A-133 to V-147; V-134 to T-148;F-135 to S-149; A-136 to V-150; L-137 to I-151; K-138 to T-152; A-139 toW-153; R-140 to V-154; T-141 to V-155; V-142 to A-156; T-143 to V-157;F-144 to F-158; G-145 to A-159; V-146 to S-160; V-147 to L-161; T-148 toP-162; S-149 to G-163; V-150 to I-164; I-151 to I-165; T-152 to F-166;W-153 to T-167; V-154 to R-168; V-155 to S-169; A-156 to Q-170; V-157 toK-171; F-158 to E-172; A-159 to G-173; S-160 to L-174; L-161 to H-175;P-162 to Y-176; G-163 to T-177; I-164 to C-178; I-165 to S-179; F-166 toS-180; T-167 to H-181; R-168 to F-182; S-169 to P-183; Q-170 to Y-184;K-171 to S-185; E-172 to Q-186; G-173 to Y-187; L-174 to Q-188; H-175 toF-189; Y-176 to W-190; T-177 to K-191; C-178 to N-192; S-179 to F-193;S-180 to Q-194; H-181 to T-195; F-182 to L-196; P-183 to K-197; Y-184 toI-198; S-185 to V-199; Q-186 to 1-200; Y-187 to L-201; Q-188 to G-202;F-189 to L-203; W-190 to V-204; K-191 to L-205; N-192 to P-206; F-193 toL-207; Q-194 to L-208; T-195 to V-209; L-196 to M-210; K-197 to V-211;I-198 to I-212; V-199 to C-213; I-200 to Y-214; L-201 to S-215; G-202 toG-216; L-203 to I-217; V-204 to L-218; L-205 to K-219; P-206 to T-220;L-207 to L-221; L-208 to L-222; V-209 to R-223; M-210 to C-224; V-211 toR-225; I-212 to N-226; C-213 to E-227; Y-214 to K-228; S-215 to K-229;G-216 to R-230; I-217 to H-231; L-218 to R-232; K-219 to A-233; T-220 toV-234; L-221 to R-235; L-222 to L-236; R-223 to I-237; C-224 to F-238;R-225 to T-239; N-226 to I-240; E-227 to M-241; K-228 to I-242; K-229 toV-243; R-230 to Y-244; H-231 to F-245; R-232 to L-246; A-233 to F-247;V-234 to W-248; R-235 to A-249; L-236 to P-250; I-237 to Y-251; F-238 toN-252; T-239 to I-253; I-240 to V-254; M-241 to L-255; I-242 to L-256;V-243 to L-257; Y-244 to N-258; F-245 to T-259; L-246 to F-260; F-247 toQ-261; W-248 to E-262; A-249 to F-263; P-250 to F-264; Y-251 to G-265;N-252 to L-266; I-253 to N-267; V-254 to N-268; L-255 to C-269; L-256 toS-270; L-257 to S-271; N-258 to S-272; T-259 to N-273; F-260 to R-274;Q-261 to L-275; E-262 to D-276; F-263 to Q-277; F-264 to A-278; G-265 toM-279; L-266 to Q-280; N-267 to V-281; N-268 to T-282; C-269 to E-283;S-270 to T-284; S-271 to L-285; S-272 to G-286; N-273 to M-287; R-274 toT-288; L-275 to H-289; D-276 to C-290; Q-277 to C-291; A-278 to I-292;M-279 to N-293; Q-280 to P-294; V-281 to I-295; T-282 to 1-296; E-283 toY-297; T-284 to A-298; L-285 to F-299; G-286 to V-300; M-287 to G-301;T-288 to E-302; H-289 to K-303; C-290 to F-304; C-291 to R-305; I-292 toN-306; N-293 to Y-307; P-294 to L-308; I-295 to L-309; I-296 to V-310;Y-297 to F-311; A-298 to F-312; F-299 to Q-313; V-300 to K-314; G-301 toH-315; E-302 to I-316; K-303 to A-317; F-304 to K-318; R-305 to R-319;N-306 to F-320; Y-307 to C-321; L-308 to K-322; L-309 to C-323; V-310 toC-324; F-311 to S-325; F-312 to I-326; Q-313 to F-327; K-314 to Q-328;H-315 to Q-329; I-316 to E-330; A-317 to A-331; K-318 to P-332; R-319 toE-333; F-320 to R-334; C-321 to A-335; K-322 to S-336; C-323 to S-337;C-324 to V-338; S-325 to Y-339; I-326 to T-340; F-327 to R-341; Q-328 toS-342; Q-329 to T-343; E-330 to E-344; A-331 to E-345; P-332 to Q-346;E-333 to E-347; R-334 to I-348; A-335 to S-349; S-336 to V-350; S-337 toG-351; and/or V-338 to L-352 of SEQ ID NO:22.

These polypeptide fragments may retain the biological activity ofG-protein Chemokine Receptor (CCR5) polypeptides of the invention and/ormay be useful to generate or screen for antibodies, as described furtherbelow. Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the G-protein Chemokine Receptor (CCR5)polypeptide described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence.

Additionally, the present application is also directed to proteinscontaining polypeptides at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to the G-protein Chemokine Receptor (CCR5) polypeptidefragments set forth above. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a G-protein Chemokine Receptor (CCR5)functional activity. By a polypeptide demonstrating a G-proteinChemokine Receptor (CCR5) “functional activity” is meant, a polypeptidecapable of displaying one or more known functional activities associatedwith a full-length (complete) G-protein Chemokine Receptor (CCR5)protein. Such functional activities include, but are not limited to,biological activity, antigenicity [ability to bind (or compete with aG-protein Chemokine Receptor (CCR5) polypeptide for binding) to ananti-G-protein Chemokine Receptor (CCR5) antibody], immunogenicity(ability to generate antibody which binds to a G-protein ChemokineReceptor (CCR5) polypeptide), ability to form multimers with G-proteinChemokine Receptor (CCR5) polypeptides of the invention, and ability tobind to a receptor or ligand for a G-protein Chemokine Receptor (CCR5)polypeptide.

The functional activity of G-protein Chemokine Receptor (CCR5)polypeptides, and fragments, variants derivatives, and analogs thereof,can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length G-protein Chemokine Receptor (CCR5)polypeptide for binding to anti-G-protein Chemokine Receptor (CCR5)antibody, various immunoassays known in the art can be used, includingbut not limited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a G-protein Chemokine Receptor (CCR5)ligand is identified, or the ability of a polypeptide fragment, variantor derivative of the invention to multimerize is being evaluated,binding can be assayed, e.g., by means well-known in the art, such as,for example, reducing and non-reducing gel chromatography, proteinaffinity chromatography, and affinity blotting. See generally, Phizicky,E., et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment,physiological correlates of G-protein Chemokine Receptor (CCR5) bindingto its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability of G-proteinChemokine Receptor (CCR5) polypeptides and fragments, variantsderivatives and analogs thereof to elicit G-protein Chemokine Receptor(CCR5) related biological activity (either in vitro or in vivo). Othermethods will be known to the skilled artisan and are within the scope ofthe invention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of G-proteinChemokine Receptor. Such fragments include amino acid residues thatcomprise alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet-forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) G-proteinChemokine Receptor (CCR5) (SEQ ID NO:2) or encoded by the depositedclone. Certain preferred regions are those set out in FIG. 3 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIG. 1(SEQ ID NO:2) or encoded by the deposited clone, such preferred regionsinclude; Garnier-Robson predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions,beta-regions, turn-regions, and coil-regions; Kyte-Doolittle predictedhydrophilic and hydrophobic regions; Eisenberg alpha and betaamphipathic regions; Emini surface-forming regions; and Jameson-Wolfhigh antigenic index regions, as predicted using the default parametersof these computer programs. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of G-protein Chemokine Receptor. Preferredembodiments of the invention in this regard include fragments thatcomprise alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of G-proteinChemokine Receptor.

The data representing the structural or functional attributes ofG-protein Chemokine Receptor (CCR5) set forth in FIG. 1 or encoded bythe deposited clone and/or Table 1, as described above, was generatedusing the various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, IX, XIII, and XIV of Table 1 can be used to determine regions ofG-protein Chemokine Receptor (CCR5) which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or IV by choosingvalues which represent regions of the polypeptide which are likely to beexposed on the surface of the polypeptide in an environment in whichantigen recognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table 1, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table 1). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table 1include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 1 orencoded by the deposited clone. As set out in FIG. 3 and in Table 1,such preferred regions include Garnier-Robson alpha-regions,beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions,beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions andhydrophobic regions, Eisenberg alpha- and beta-amphipathic regions,Karplus-Schulz flexible regions, Emini surface-forming regions andJameson-Wolf regions of high antigenic index.

TABLE 1 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 . . B . . . . 0.24 0.06 . * . 0.05 1.68 Asp 2 . . B B . . . 0.33 0.27. * . −0.30 0.98 Tyr 3 . . B B . . . 0.42 0.23 . * . −0.15 1.02 Gln 4 .. B B . . . 0.60 0.19 . * . −0.15 1.38 Val 5 . . B B . . . 0.10 0.00 . *F 0.00 1.28 Ser 6 . . B B . . . 0.46 0.69 * * F −0.45 0.57 Ser 7 . . B B. . . 0.46 0.69 * * F −0.45 0.52 Pro 8 . . B B . . . −0.19 0.29 * * F0.00 1.17 Ile 9 . . B B . . . −0.19 0.33 * * . −0.30 0.61 Tyr 10 . . B B. . . 0.42 0.34 * . . −0.30 0.73 Asp 11 . . B . . T . 0.48 0.71 . . .−0.20 0.74 Ile 12 . . B . . T . 0.47 1.04 . . . −0.05 1.66 Asn 13 . . B. . T . 0.38 0.84 * . . −0.05 1.53 Tyr 14 . . B . . T . 1.27 0.47 * . .−0.05 1.23 Tyr 15 . . . B T . . 1.30 0.47 . * . −0.05 3.03 Thr 16 . . .B T . . 0.63 0.21 . * F 0.65 2.91 Ser 17 . . . B T . . 1.31 0.39 . * F0.75 1.00 Glu 18 . . B . . . . 1.36 0.06 . * F 0.80 0.98 Pro 19 . . . .T . . 0.71 −0.70 . * F 2.50 1.36 Cys 20 . . . . T T . 0.96 −0.50 . * F2.50 0.71 Pro 21 . . . . T T . 0.41 −0.49 . * F 2.25 0.66 Lys 22 . . . .T T . 0.76 0.16 . * F 1.40 0.32 Ile 23 A . . . . T . 0.76 −0.27 * * F1.50 1.19 Asn 24 A A . . . . . 0.08 −0.44 . * F 0.85 1.33 Val 25 A A . .. . . 0.16 −0.19 . * . 0.30 0.47 Lys 26 . A B . . . . −0.22 0.31 . * .−0.30 0.67 Gln 27 . A B . . . . −0.16 0.13 * * . −0.30 0.42 Ile 28 . A B. . . . −0.08 −0.27 * * . 0.45 1.11 Ala 29 . A B . . . . −0.89 −0.23 * *. 0.30 0.46 Ala 30 . A B . . . . −0.24 0.46 * * . −0.60 0.22 Arg 31 . AB . . . . −0.50 0.49 * * . −0.60 0.48 Leu 32 . A B . . . . −1.310.23 * * . −0.30 0.74 Leu 33 . A B . . . . −0.67 0.41 * * . −0.60 0.60Pro 34 . . . . . T C −0.38 0.67 * * F 0.15 0.48 Pro 35 . . . . T T .−0.60 1.06 * * F 0.35 0.78 Leu 36 . . B . . T . −1.57 1.06 * * . −0.200.78 Tyr 37 . . B . . T . −1.46 1.01 . . . −0.20 0.38 Ser 38 . . B B . .. −1.53 1.37 . . . −0.60 0.21 Leu 39 . . B B . . . −2.02 1.63 . . .−0.60 0.18 Val 40 . . B B . . . −2.16 1.73 . . . −0.60 0.10 Phe 41 . . BB . . . −2.04 1.40 . . . −0.60 0.07 Ile 42 . . B B . . . −2.66 1.80 . .. −0.60 0.08 Phe 43 . . B B . . . −2.70 1.76 . . . −0.60 0.08 Gly 44 . .B B . . . −1.89 1.54 . . . −0.60 0.09 Phe 45 . . . B T . . −1.63 1.16 .. . −0.20 0.20 Val 46 . . . B . . C −1.74 1.09 . . . −0.40 0.23 Gly 47 .. . B . . C −1.71 0.99 . . . −0.40 0.19 Asn 48 A . . B . . . −1.90 1.20. . . −0.60 0.16 Met 49 A . . B . . . −2.37 1.10 . . . −0.60 0.15 Leu 50A . . B . . . −2.56 1.14 . . . −0.60 0.13 Val 51 . . B B . . . −2.511.40 . . . −0.60 0.06 Ile 52 . . B B . . . −3.06 1.69 . . . −0.60 0.05Leu 53 . . B B . . . −3.06 1.76 . . . −0.60 0.04 Ile 54 . . B B . . .−3.12 1.47 . * . −0.60 0.09 Leu 55 . . B B . . . −2.31 1.40 * . . −0.600.07 Ile 56 . . B B . . . −1.34 1.11 * . . −0.60 0.14 Asn 57 . . B B . .. −1.27 0.43 . * . −0.60 0.39 Cys 58 . A B B . . . −0.46 0.43 . . .−0.60 0.39 Gln 59 A A . B . . . 0.13 −0.26 * . . 0.30 0.96 Arg 60 A A .B . . . 0.34 −0.56 . . F 0.75 0.80 Leu 61 A A . B . . . 0.92 −0.34 . . F0.60 1.47 Glu 62 . A B B . . . 0.92 −0.43 . * F 0.60 1.23 Ser 63 . A B .. . . 0.70 −0.83 * * F 0.90 1.05 Met 64 . A B B . . . 0.46 −0.14 * * F0.45 0.89 Thr 65 . A B B . . . −0.47 −0.07 . * F 0.45 0.80 Asp 66 A A .B . . . −0.47 0.61 . * . −0.60 0.50 Ile 67 A A . B . . . −0.47 0.91 . .. −0.60 0.41 Tyr 68 A A . B . . . −0.98 0.70 . . . −0.60 0.46 Leu 69 A A. B . . . −0.97 0.90 . . . −0.60 0.23 Leu 70 A A . B . . . −1.54 1.40. * . −0.60 0.33 Asn 71 A A . B . . . −1.84 1.40 . . . −0.60 0.15 Leu 72. A B B . . . −0.96 1.03 * . . −0.60 0.24 Ala 73 A A . B . . . −1.520.34 * . . −0.30 0.48 Ile 74 A A . B . . . −1.41 0.34 . . . −0.30 0.25Ser 75 A A . B . . . −1.30 0.73 . * . −0.60 0.26 Asp 76 A A . B . . .−2.11 0.83 . . . −0.60 0.22 Leu 77 . A B B . . . −2.11 1.01 . . . −0.600.26 Phe 78 . A B B . . . −1.83 1.01 . . . −0.60 0.16 Phe 79 . A B B . .. −1.80 1.11 . . . −0.60 0.14 Leu 80 . A B B . . . −1.71 1.76 . * .−0.60 0.13 Leu 81 . A B B . . . −2.41 1.50 . * . −0.60 0.22 Thr 82 . A BB . . . −1.89 1.50 . * . −0.60 0.22 Val 83 . A . B . . C −1.78 1.63 . *. −0.40 0.29 Pro 84 A A . B . . . −1.11 1.44 . * . −0.60 0.35 Phe 85 A A. B . . . −0.54 1.26 . . . −0.60 0.33 Trp 86 A A . B . . . −0.32 1.53. * . −0.60 0.70 Ala 87 A A . B . . . −0.60 1.39 . . . −0.60 0.45 His 88A A . B . . . −0.33 1.46 . . . −0.60 0.53 Tyr 89 A A . B . . . −0.121.17 . . . −0.60 0.51 Ala 90 A A . B . . . 0.29 0.66 . * . −0.60 0.87Ala 91 A A . . . . . 0.58 1.07 . * . −0.60 0.67 Ala 92 A A . . . . .0.47 0.57 . * . −0.60 0.72 Gln 93 A A . . . . . 0.16 0.60 . * . −0.600.62 Trp 94 A A . . . . . 0.40 0.53 . * . −0.60 0.60 Asp 95 . . . . T T. 0.68 0.43 . * . 0.20 0.96 Phe 96 . . . . T T . 0.67 0.41 . * . 0.200.80 Gly 97 . . . . T T . 0.59 0.63 * * F 0.35 0.75 Asn 98 . . . . T T .0.59 0.29 * * F 0.65 0.24 Thr 99 . . . B T . . 0.07 0.69 * . . −0.200.48 Met 100 . . . B T . . −0.74 0.59 * . . −0.20 0.40 Cys 101 . . B B .. . −0.36 0.84 * . . −0.60 0.21 Gln 102 . . B B . . . −0.36 0.93 * . .−0.60 0.21 Leu 103 . . B B . . . −1.17 0.87 * . . −0.60 0.21 Leu 104 . .B B . . . −1.10 0.94 . . . −0.60 0.32 Thr 105 . . B B . . . −1.20 1.13 *. . −0.60 0.29 Gly 106 . . B B . . . −1.42 1.51 * . . −0.60 0.30 Leu 107. . B B . . . −1.77 1.51 . . . −0.60 0.26 Tyr 108 . . B B . . . −1.661.26 . . . −0.60 0.18 Phe 109 . . B B . . . −1.54 1.56 . . . −0.60 0.15Ile 110 . . B B . . . −1.53 1.91 . . . −0.60 0.16 Gly 111 . . B B . . .−1.53 1.61 . . . −0.60 0.14 Phe 112 . . B B . . . −1.61 1.29 . . . −0.600.16 Phe 113 . . B B . . . −2.07 1.19 . . . −0.60 0.16 Ser 114 . . . B .. C −2.07 1.29 . . . −0.40 0.14 Gly 115 . . . B . . C −2.07 1.64 . . .−0.40 0.14 Ile 116 . . . B . . C −2.61 1.54 . . . −0.40 0.11 Phe 117 . .B B . . . −2.72 1.44 . . . −0.60 0.06 Phe 118 . . B B . . . −2.83 1.74 .. . −0.60 0.05 Ile 119 . . B B . . . −2.84 2.00 . . . −0.60 0.06 Ile 120. . B B . . . −3.39 1.80 . * . −0.60 0.10 Leu 121 . . B B . . . −2.501.70 * . . −0.60 0.08 Leu 122 . . B B . . . −1.69 0.91 * . . −0.60 0.18Thr 123 A . . B . . . −1.23 0.23 * . . −0.30 0.52 Ile 124 A . . B . . .−1.16 0.30 * . . −0.30 0.98 Asp 125 A . . . . T . −0.86 0.30 * . F 0.250.98 Arg 126 A . . . . T . −0.93 0.11 * . . 0.10 0.69 Tyr 127 A . . . .T . −0.98 0.31 * . . 0.10 0.69 Leu 128 A . . . . T . −0.70 0.27 * . .0.10 0.30 Ala 129 A . . B . . . −0.40 0.77 * . . −0.60 0.21 Ile 130 A .. B . . . −1.26 1.27 * . . −0.60 0.14 Val 131 A . . B . . . −2.07 1.16 *. . −0.60 0.12 His 132 A . . B . . . −2.41 1.26 . . . −0.60 0.11 Ala 133A . . B . . . −2.41 1.26 . * . −0.60 0.15 Val 134 A . . B . . . −1.781.26 . * . −0.60 0.17 Phe 135 A . . B . . . −1.48 0.61 . * . −0.60 0.25Ala 136 A . . B . . . −0.51 0.61 . * . −0.60 0.25 Leu 137 A . . B . . .−0.79 0.11 . * . −0.30 0.65 Lys 138 A . . B . . . −1.06 −0.04 . * F 0.601.09 Ala 139 A . . B . . . −0.51 −0.19 . * F 0.45 0.80 Arg 140 A . . B .. . −0.51 −0.20 . * F 0.60 1.40 Thr 141 . . B B . . . −0.27 −0.10 . * F0.45 0.61 Val 142 . . B B . . . −0.31 0.33 . * . −0.30 0.59 Thr 143 . .B B . . . −1.21 0.47 . * . −0.60 0.23 Phe 144 . . B B . . . −0.93 1.11 .. . −0.60 0.12 Gly 145 . . B B . . . −1.34 1.11 . . . −0.60 0.23 Val 146. . B B . . . −1.89 0.86 . . . −0.60 0.21 Val 147 . . B B . . . −1.921.01 . . . −0.60 0.18 Thr 148 . . B B . . . −1.92 0.91 . . . −0.60 0.13Ser 149 . . B B . . . −1.51 0.97 * . . −0.60 0.25 Val 150 . . B B . . .−2.02 1.24 * . . −0.60 0.35 Ile 151 . . B B . . . −2.02 1.24 * . . −0.600.18 Thr 152 . . B B . . . −1.76 1.40 * . . −0.60 0.10 Trp 153 . . B B .. . −2.30 1.51 * . . −0.60 0.14 Val 154 . . B B . . . −2.70 1.51 * . .−0.60 0.14 Val 155 . . B B . . . −2.43 1.61 * . . −0.60 0.09 Ala 156 . .B B . . . −1.84 1.63 * . . −0.60 0.08 Val 157 . . B B . . . −2.34 1.10 .. . −0.60 0.15 Phe 158 . . B B . . . −2.27 1.14 . . . −0.60 0.17 Ala 159. . B B . . . −1.76 0.93 . . . −0.60 0.25 Ser 160 . . . B . . C −1.790.86 . . . −0.40 0.34 Leu 161 . . . B . . C −2.09 0.90 . . . −0.40 0.27Pro 162 . . . B . . C −1.93 0.80 . . . −0.40 0.19 Gly 163 . . . B T . .−1.54 1.09 * . . −0.20 0.12 Ile 164 . . B B . . . −0.84 1.19 . . . −0.600.22 Ile 165 . . B B . . . −0.84 0.50 . * . −0.60 0.27 Phe 166 . . B B .. . −0.03 0.46 . . . −0.26 0.37 Thr 167 . . B . . T . 0.22 0.43 * . F0.63 0.91 Arg 168 . . B . . T . 0.57 −0.26 * . F 2.02 2.60 Ser 169 . . .. . T C 1.11 −0.94 * . F 2.86 5.21 Gln 170 . . . . T T . 1.19 −1.30 * .F 3.40 3.57 Lys 171 . . . . T . . 1.86 −1.10 * . F 2.86 1.50 Glu 172 . .. . T . . 1.92 −0.60 * . F 2.52 1.53 Gly 173 . . . B T . . 1.50−0.23 * * F 1.68 1.38 Leu 174 . . B B . . . 1.13 −0.14 . . . 0.64 1.00His 175 . . B B . . . 0.83 0.43 . . . −0.60 0.31 Tyr 176 . . B B . . .0.49 0.81 . . . −0.60 0.42 Thr 177 . . B B . . . 0.46 0.77 . * . −0.600.68 Cys 178 . . B . . T . 0.10 0.59 . * . −0.20 0.68 Ser 179 . . . . TT . 0.70 0.87 . * . 0.20 0.38 Ser 180 . . . . T T . 0.49 0.54 . . . 0.200.40 His 181 . . . . T T . 0.43 0.81 . . . 0.35 1.18 Phe 182 . . . . . TC 0.74 0.63 . . . 0.15 1.18 Pro 183 . . . . T T . 1.17 0.64 . . . 0.351.52 Tyr 184 . . . . T T . 1.47 1.01 . * . 0.35 1.75 Ser 185 . . . . T T. 1.07 0.91 . . . 0.35 3.50 Gln 186 . . B B . . . 0.81 0.91 * . . −0.451.96 Tyr 187 . . . B T . . 1.56 1.40 * . . −0.05 1.31 Gln 188 . . . B T. . 1.77 0.64 * . . −0.05 1.96 Phe 189 . . . B T . . 1.31 0.66 * * .−0.05 1.82 Trp 190 . . . B T . . 1.61 1.04 * * . −0.05 1.01 Lys 191 . .B B . . . 1.30 0.69 * * . −0.45 1.01 Asn 192 . . . B T . . 0.73 0.77 * .. −0.05 1.68 Phe 193 . . . B T . . 0.78 0.67 * * . −0.05 1.32 Gln 194 A. . B . . . 0.59 −0.24 * . F 0.60 1.32 Thr 195 . . . B . . C 0.020.44 * * F −0.25 0.57 Leu 196 . . B B . . . −0.91 0.69 * . . −0.60 0.49Lys 197 . . B B . . . −1.72 0.59 . . . −0.60 0.20 Ile 198 . . B B . . .−1.37 0.87 * . . −0.60 0.11 Val 199 . . B B . . . −2.18 0.81 * * . −0.600.14 Ile 200 . . B B . . . −2.72 0.81 . * . −0.60 0.06 Leu 201 . . B B .. . −2.72 1.46 . * . −0.60 0.06 Gly 202 . . B B . . . −2.98 1.46 . * .−0.60 0.07 Leu 203 . . B B . . . −2.90 1.24 . . . −0.60 0.15 Val 204 . .B B . . . −2.86 1.24 . . . −0.60 0.15 Leu 205 . . B B . . . −2.82 1.24 .. . −0.60 0.12 Pro 206 . . B B . . . −2.61 1.46 . . . −0.60 0.11 Leu 207. . B B . . . −3.12 1.39 . . . −0.60 0.15 Leu 208 . . B B . . . −3.201.39 . . . −0.60 0.13 Val 209 . . B B . . . −3.01 1.39 . . . −0.60 0.06Met 210 . . B B . . . −2.44 1.53 . . . −0.60 0.04 Val 211 . . B B . . .−2.53 1.60 . . . −0.60 0.07 Ile 212 . . B B . . . −2.07 1.30 . . . −0.600.13 Cys 213 . . B . . T . −2.14 1.09 . . . −0.20 0.13 Tyr 214 . . B . .T . −2.10 1.16 . . . −0.20 0.13 Ser 215 . . B . . T . −1.46 1.20 . . .−0.20 0.15 Gly 216 . . B . . T . −0.91 0.51 * . . −0.20 0.55 Ile 217 . .B B . . . −0.83 0.43 . . . −0.60 0.51 Leu 218 . . B B . . . −0.980.36 * * . −0.30 0.31 Lys 219 . . B B . . . −0.62 0.66 * * F −0.45 0.26Thr 220 . . B B . . . −0.99 0.23 * * . −0.30 0.73 Leu 221 . . B B . . .−0.53 0.11 * * . −0.30 0.47 Leu 222 A . . B . . . 0.36 −0.57 * * . 0.600.46 Arg 223 A . . B . . . 1.17 −0.17 * * . 0.30 0.52 Cys 224 A . . . .T . 1.17 −0.66 . * . 1.15 1.08 Arg 225 A . . . . T . 1.52 −1.34 * * F1.30 2.63 Asn 226 A . . . . T . 2.44 −2.03 * * F 1.30 2.68 Glu 227 A . .. . T . 3.22 −2.03 . * F 1.30 9.80 Lys 228 A . . . . . . 3.22 −2.10 . *F 1.10 6.81 Lys 229 A . . . . . . 3.30 −2.10 * * F 1.10 8.29 Arg 230 A .. . . . . 2.33 −2.00 * . F 1.10 4.83 His 231 A . . B . . . 2.44−1.36 * * . 0.75 1.79 Arg 232 A . . B . . . 1.63 −1.36 * . . 0.75 1.76Ala 233 A . . B . . . 0.70 −0.67 * * . 0.60 0.74 Val 234 A . . B . . .−0.04 0.01 * . . −0.30 0.38 Arg 235 A . . B . . . −0.47 0.30 * * . −0.300.17 Leu 236 . . B B . . . −1.32 0.79 * * . −0.60 0.24 Ile 237 . . B B .. . −2.03 0.97 * * . −0.60 0.23 Phe 238 . . B B . . . −2.33 0.94 * * .−0.60 0.11 Thr 239 . . B B . . . −2.33 1.63 * * . −0.60 0.10 Ile 240 . .B B . . . −2.69 1.59 * * . −0.60 0.10 Met 241 . . B B . . . −2.58 1.66 .. . −0.60 0.19 Ile 242 . . B B . . . −2.50 1.66 . . . −0.60 0.11 Val 243. . B B . . . −2.50 1.86 . . . −0.60 0.13 Tyr 244 . . B B . . . −2.481.96 . . . −0.60 0.12 Phe 245 . . B B . . . −2.18 2.26 . . . −0.60 0.17Leu 246 . . B B . . . −1.79 2.07 . . . −0.60 0.24 Phe 247 . . . B T . .−1.14 1.86 . . . −0.20 0.23 Trp 248 . . . B . . C −0.29 1.86 . * . −0.400.42 Ala 249 . . . . . T C −0.93 1.47 . . . 0.00 0.82 Pro 250 . . . . .T C −1.09 1.47 . * . 0.00 0.67 Tyr 251 . . . . T T . −1.09 1.33 . . .0.20 0.47 Asn 252 . . B . . T . −1.20 1.10 . * . −0.20 0.38 Ile 253 . .B B . . . −1.72 1.29 . * . −0.60 0.20 Val 254 . . B B . . . −1.13 1.54. * . −0.60 0.11 Leu 255 . . B B . . . −1.23 1.19 * . . −0.60 0.11 Leu256 . . B B . . . −1.69 1.27 * . . −0.60 0.22 Leu 257 . . B B . . .−1.69 1.37 * . . −0.60 0.26 Asn 258 . . B B . . . −0.80 1.13 * . . −0.600.54 Thr 259 A . . B . . . −0.64 0.44 * . . −0.45 1.14 Phe 260 A . . B .. . −0.53 0.54 * . . −0.45 1.20 Gln 261 . . B B . . . −0.07 0.64 * . .−0.60 0.64 Glu 262 . . B B . . . −0.07 0.67 * . . −0.60 0.44 Phe 263 . .B . . . . −0.07 0.87 * . . −0.40 0.42 Phe 264 . . . . T . . 0.24 0.49 .. . 0.00 0.39 Gly 265 . . . . T . . 0.28 0.49 . . . 0.00 0.36 Leu 266 .. . . T . . −0.02 1.06 . . . 0.00 0.22 Asn 267 . . . . T . . −0.32 0.66. . . 0.00 0.35 Asn 268 . . . . T . . 0.08 0.26 . . F 0.45 0.47 Cys 269. . . . T T . 0.78 0.21 * * F 0.65 0.76 Ser 270 . . . . T T . 1.23−0.07 * * F 1.25 0.76 Ser 271 . . . . T T . 1.23 −0.47 . * F 1.25 0.93Ser 272 . . . . . T C 1.23 −0.19 * * F 1.20 1.43 Asn 273 . A . . T . .1.23 −0.76 . * F 1.30 1.79 Arg 274 . A . . T . . 1.31 −0.74 * * F 1.302.31 Leu 275 A A . . . . . 1.01 −0.63 * * F 0.90 1.74 Asp 276 A A . . .. . 1.31 −0.40 * * F 0.60 1.07 Gln 277 A A . . . . . 0.76 −0.40 * * .0.30 0.95 Ala 278 A . . B . . . 0.44 0.24 * * . −0.30 0.85 Met 279 . . BB . . . 0.33 0.04 * * . −0.30 0.74 Gln 280 . . B B . . . 0.83 0.04 * * .−0.30 0.74 Val 281 . . B B . . . 0.02 0.13 * . . −0.15 1.05 Thr 282 A .. B . . . −0.32 0.31 * . F −0.15 0.88 Glu 283 A . . B . . . −0.33 0.13 *. F −0.15 0.50 Thr 284 A . . B . . . −0.04 0.34 . * F −0.15 0.67 Leu 285A . . B . . . −0.08 0.19 . . . −0.30 0.67 Gly 286 . . . B T . . 0.110.20 . . . 0.10 0.52 Met 287 . . . B T . . −0.24 0.77 . . . −0.20 0.19Thr 288 . . B B . . . −1.13 0.86 . . . −0.60 0.13 His 289 . . B B . . .−0.82 0.86 . . . −0.60 0.09 Cys 290 . . B B . . . −0.22 0.83 . . . −0.600.15 Cys 291 . . B B . . . −0.77 0.64 . . . −0.60 0.16 Ile 292 . . B B .. . −1.06 0.84 . . . −0.60 0.08 Asn 293 . . B B . . . −0.99 1.03 * * .−0.60 0.11 Pro 294 . . B B . . . −1.54 1.21 * . . −0.60 0.31 Ile 295 . AB B . . . −1.58 1.14 * . . −0.60 0.44 Ile 296 . A B B . . . −1.77 1.24 *. . −0.60 0.24 Tyr 297 . A B B . . . −1.22 1.49 * . . −0.60 0.11 Ala 298. A B B . . . −1.22 1.49 . . . −0.60 0.16 Phe 299 . A B B . . . −0.970.80 . . . −0.60 0.40 Val 300 . A B B . . . −0.78 0.11 * * . −0.30 0.51Gly 301 A A . B . . . 0.22 0.14 * * F −0.15 0.44 Glu 302 A A . . . . .0.47 −0.36 * * F 0.45 0.99 Lys 303 A A . . . . . 0.81 −0.74 * * F 0.902.14 Phe 304 A . . . . T . 0.70 −0.63 * * F 1.30 3.39 Arg 305 A . . . .T . 0.74 −0.37 * * . 0.85 1.62 Asn 306 A . . . . T . 0.23 0.31 * * .0.10 0.67 Tyr 307 A . . . . T . −0.47 0.96 * * . −0.20 0.57 Leu 308 A .. B . . . −1.21 0.96 * * . −0.60 0.25 Leu 309 A . . B . . . −0.511.74 * * . −0.60 0.14 Val 310 A . . B . . . −0.58 1.74 . . . −0.60 0.15Phe 311 A . . B . . . −0.61 0.99 * . . −0.60 0.36 Phe 312 A . . B . . .−1.26 0.80 * . . −0.60 0.60 Gln 313 A . . B . . . −1.03 0.80 * . . −0.600.57 Lys 314 A . . B . . . −0.18 0.66 * . . −0.60 0.66 His 315 A A . . .. . 0.79 −0.13 * * . 0.45 1.53 Ile 316 A A . . . . . 0.79 −0.91 * . .0.75 1.73 Ala 317 A A . . . . . 0.82 −0.53 * . . 0.88 0.75 Lys 318 A A .. . . . 0.87 0.04 * . . 0.26 0.30 Arg 319 . A . . T . . 0.16 −0.46 * . .1.54 0.84 Phe 320 . A . . T . . −0.48 −0.57 * . . 2.12 0.45 Cys 321 . .. . T T . 0.11 −0.50 * . . 2.80 0.12 Lys 322 . . . . T T . −0.19 −0.11 *. . 2.22 0.08 Cys 323 . . . . T T . −0.93 0.57 * . . 1.04 0.07 Cys 324 .. . . T T . −1.04 0.57 * . . 0.76 0.11 Ser 325 . A . . T . . −0.340.40 * . . 0.38 0.09 Ile 326 . A B . . . . 0.32 0.80 * . . −0.60 0.30Phe 327 . A B . . . . −0.31 0.23 * . . −0.30 0.97 Gln 328 A A . . . . .0.14 0.16 . . F −0.15 0.73 Gln 329 A A . . . . . 0.81 0.20 . * F 0.001.61 Glu 330 A A . . . . . 1.22 −0.49 . * F 0.60 3.22 Ala 331 . A . . .. C 1.52 −1.27 . * F 1.10 3.64 Pro 332 A A . . . . . 1.92 −1.17 * * F0.90 2.13 Glu 333 A A . . . . . 1.62 −1.19 * * F 0.90 1.64 Arg 334 A A .. . . . 0.77 −0.80 * . F 0.90 2.18 Ala 335 A A . B . . . 0.52 −0.66 * *F 0.90 1.05 Ser 336 . A B B . . . 0.80 −0.33 * * F 0.45 0.95 Ser 337 . .B B . . . 1.12 0.16 * . F −0.15 0.70 Val 338 . . B B . . . 0.82 0.16 * *. −0.15 1.35 Tyr 339 . . B B . . . 0.40 0.04 * * F 0.30 1.35 Thr 340 . .B B . . . 0.64 0.14 * * F 0.60 1.46 Arg 341 . . . B . . C 0.94 0.19 . *F 1.10 1.94 Ser 342 . . . . . T C 1.24 −0.46 . * F 2.40 2.15 Thr 343 . .. . . T C 2.10 −0.81 . * F 3.00 2.58 Gly 344 . . . . . T C 1.46 −1.30. * F 2.70 2.28 Glu 345 . . . . . T C 1.47 −0.61 . * F 2.40 1.19 Gln 346. . B B . . . 0.50 −0.61 . * F 1.50 1.11 Glu 347 . . B B . . . 0.46−0.46 . * F 0.75 0.83 Ile 348 . . B B . . . −0.04 −0.46 . * F 0.45 0.47Ser 349 . . B B . . . −0.09 0.23 . * . −0.30 0.23 Val 350 . . B B . . .−0.48 0.26 . * . −0.30 0.17 Gly 351 . . B B . . . −0.87 0.69 . * . −0.600.30 Leu 352 A . . B . . . −1.26 0.43 . * . −0.60 0.29

Among highly preferred fragments in this regard are those that compriseregions of G-protein Chemokine Receptor (CCR5) that combine severalstructural features, such as several of the features set out above.

Other preferred polypeptide fragments are biologically active G-proteinChemokine Receptor (CCR5) fragments. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the G-protein Chemokine Receptor (CCR5) polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity. Polynucleotides encodingthese polypeptide fragments are also encompassed by the invention.

However, many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 or to the deposited clone andmay have been publicly available prior to conception of the presentinvention. Preferably, such related polynucleotides are specificallyexcluded from the scope of the present invention. To list every relatedsequence would be cumbersome. Accordingly, preferably excluded from thepresent invention are one or more polynucleotides comprising anucleotide sequence described by the general formula of a-b, where a isany integer between 1 to 1400 of SEQ ID NO:1, b is an integer of 15 to1414, where both a and b correspond to the positions of nucleotideresidues shown in SEQ ID NO:1 or of the deposited clone, and where the bis greater than or equal to a +14.

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC DepositNo: 97183 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:1 or contained in ATCC DepositNo: 97183 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:1 or the sequence of the depositedclone), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic. Either the full-lengthprotein or an antigenic peptide fragment can be used. Regions having ahigh antigenicity index are shown in Table 1 and FIG. 3.

Antibodies are preferably prepared from these regions or from discretefragments in these regions. However, antibodies can be prepared from anyregion of the peptide as described herein. A preferred fragment producesan antibody that diminishes or completely prevents ligand binding.Antibodies can be developed against the entire receptor or portions ofthe receptor, for example, the intracellular carboxy terminal domain,the amino terminal extracellular domain, the entire transmembrane domainor specific transmembrane segments, any of the intracellular orextracellular loops, or any portions of these regions. Antibodies mayalso be developed against specific functional sites, such as the site ofligand binding, the site of G-protein coupling, or sites that areglycosylated, phosphorylated, myristoylated, or amidated.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).These fragments are not to be construed, however, as encompassing anyfragments which may be disclosed prior to the invention.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs), first described by J. P. Tam in Proc.Natl. Acad. Sci. U.S.A. 85:5409 which is incorporated by referenceherein in its entirety. MAPs consist of multiple copies of a specificpeptide attached to a non-immunogenic lysine core. Map peptides usuallycontain four or eight copies of the peptide often referred to as MAP-4or MAP-8 peptides. By way of non-limiting example, MAPs may besynthesized onto a lysine core matrix attached to a polyethyleneglycol-polystyrene (PEG-PS) support. The peptide of interest issynthesized onto the lysine residues using 9-fluorenylmethoxycarbonyl(Fmoc) chemistry. For example, Applied Biosystems (Foster City, Calif.)offers MAP resins, such as, for example, the Fmoc Resin 4 Branch and theFmoc Resin 8 Branch which can be used to synthesize MAPs. Cleavage ofMAPs from the resin is performed with standard trifloroacetic acid(TFA)-based cocktails known in the art. Purification of MAPs, except fordesalting, is not necessary. MAP peptides may be used as an immunizingvaccine which elicits antibodies that recognize both the MAP and thenative protein from which the peptide was derived.

Epitope bearing peptides of the invention may also be incorporated intoa coat protein of a virus which can then be used as an immunogen or avaccine with which to immunize animals, including humans, in orderencourage the production of anti-epitope antibodies. For example, the V3loop of the gp120 glycoprotein of the human immunodeficiency virus type1 (HIV-1) has been engineered to be expressed on the surface ofrhinovirus. Immunization with this rhinovirus displaying the V3 looppeptide yielded apparently effective mimics of the HIV-1 immunogens (asmeasured by their ability to be neutralized by anti-HIV-1 antibodies aswell as their ability to elicit the production of antibodies capable ofneutralizing HIV-1 in cell culture). This techniques of using engineeredviral particles as an immunogen is described in more detail in Smith etal., Behring Inst Mitt Feb;(98):229-39 (1997), Smith et al, J Virol72:651-9 (1998), and Zhang et al., Biol Chem 380:365-74 (1999), whichare hereby incorporated by reference herein in their entireties.

Epitope bearing polypeptides of the invention may be modified, forexample, by the addition of amino acids at the amino- and/orcarboxy-termini of the peptide. Such modifications may be performed, forexample, to alter the conformation of the epitope bearing polypeptidesuch that the epitope will have a conformation more closely related tothe structure of the epitope in the native protein. An example of amodified epitope-bearing polypeptide of the invention is a polypeptidein which one or more cysteine residues have been added to thepolypeptide to allow for the formation of a disulfide bond between twocysteines, resulting in a stable loop structure of the epitope bearingpolypeptide under non-reducing conditions. Disulfide bonds may formbetween a cysteine residue added to the polypeptide and a cysteineresidue of the naturally occurring epitope, or may form between twocysteines which have both been added to the naturally occurring epitopebearing polypeptide. Additionally, it is possible to modify one or moreamino acid residues of the naturally occurring epitope bearingpolypeptide by substituting them with cysteines to promote the formationof disulfide bonded loop structures. Cyclic thioether molecules ofsynthetic peptides may be routinely generated using techniques known inthe art and are described in PCT publication WO 97/46251, incorporatedin its entirety by reference herein. Other modifications ofepitope-bearing polypeptides contemplated by this invention includebiotinylation.

Animals such as rabbits, rats and mice are immunized with either free orcarrier-coupled peptides or MAP peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)or albumin (including but not limited to recombinant human albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)), resulting in chimeric polypeptides. Such fusion proteins mayfacilitate purification and may increase half-life in vivo. This hasbeen shown for chimeric proteins consisting of the first two domains ofthe human CD4-polypeptide and various domains of the constant regions ofthe heavy or light chains of mammalian immunoglobulins. See, e.g., EP394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced deliveryof an antigen across the epithelial barrier to the immune system hasbeen demonstrated for antigens (e.g., insulin) conjugated to an FcRnbinding partner such as IgG or Fc fragments (see, e.g., PCT PublicationsWO 96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2 or of the polypeptideencoded by the deposited clone, and/or an epitope, of the presentinvention (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding).

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe heavy and the light chains of each pair are aligned by the frameworkregions, enabling binding to a specific epitope. From N-terminal toC-terminal, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547 1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J. 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992)).

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), intracellularly-made antibodies (i.e., intrabodies), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions or fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.In a preferred embodiment, the immunoglobulin is an IgM isotype. In apreferred embodiment, the immunoglobulin is an IgG1 isotype. In anotherpreferred embodiment, the immunoglobulin is an IgG2 isotype. In anotherpreferred embodiment, the immunoglobulin is an IgG4 isotype.Immunoglobulins may have both a heavy and light chain. An array of IgG,IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a lightchain of the kappa or lambda forms.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Preferred epitopes of theinvention include: Thr16-Val25, Gln59-Thr65, Thr167-Leu174, Ser179-Ser185, Leu222-Ala233, Asn268-Gln277, His315-Ser325, Glu330-Ser336,Tyr339-Ile348 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone, as well as polynucleotides that encode these epitopes.Even more preferred epitopes of the invention include peptidescorresponding the extracellular loops of the G-protein ChemokineReceptor (CCR5) of the invention or fragments and variants thereof,e.g., amino acids 89-102, 167-195 and/or 261-274 of SEQ ID NO:2 or ofthe polypeptide encoded by the deposited clone. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, monkey, rat and/or rabbit homologs of humanproteins and the corresponding epitopes thereof. Antibodies that do notbind polypeptides with less than 95%, less than 90%, less than 85%, lessthan 80%, less than 75%, less than 70%, less than 65%, less than 60%,less than 55%, and less than 50% identity (as calculated using methodsknown in the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein).

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) maybind immunospecifically to that immunospecifically bind to a polypeptideor polypeptide fragment or variant of human G-protein Chemokine Receptor(CCR5) (SEQ ID NO:2 or of the polypeptide encoded by the depositedclone) and/or monkey G-protein Chemokine Receptor (CCR5). Preferably,the antibodies of the invention bind immunospecifically to humanG-protein Chemokine Receptor. Preferably, the antibodies of theinvention bind immunospecifically to human and monkey G-proteinChemokine Receptor. Also preferably, the antibodies of the inventionbind immunospecifically to human G-protein Chemokine Receptor (CCR5) andmurine G-protein Chemokine Receptor. More preferably, antibodies of theinvention, bind immunospecifically and with higher affinity to humanG-protein Chemokine Receptor (CCR5) than to murine G-protein ChemokineReceptor.

In preferred embodiments, the antibodies of the present invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof), immunospecifically bind toG-protein Chemokine Receptor (CCR5) and do not cross-react with anyother antigens. In preferred embodiments, the antibodies of theinvention immunospecifically bind to G-protein Chemokine Receptor (CCR5)and do not cross-react with other chemokine receptors such as, forexample, US28, CCR1, CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CXCR1,CXCR2, CXCR3, CXCR4, and/or CXCR5.

In other preferred embodiments, the antibodies of the inventionimmunospecifically bind to G-protein Chemokine Receptor (CCR5) andcross-react with other chemokine receptors such as, for example, US28,CCR1, CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR3,CXCR4, and/or CXCR5. In more preferred embodiments, the antibodies ofthe invention immunospecifically bind to G-protein Chemokine Receptor(CCR5) and do cross-react with CCR3 and/or CXCR4.

In a preferred embodiment, antibodies of the invention preferentiallybind G-protein Chemokine Receptor (CCR5) (SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone), or fragments and variantsthereof relative to their ability to bind other antigens, (such as, forexample, other chemokine receptors).

By way of non-limiting example, an antibody may be considered to bind afirst antigen preferentially if it binds said first antigen with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second antigen. In another non-limiting embodiment, an antibody maybe considered to bind a first antigen preferentially if it binds saidfirst antigen with an affinity that is at least one order of magnitudeless than the antibody's K_(D) for the second antigen. In anothernon-limiting embodiment, an antibody may be considered to bind a firstantigen preferentially if it binds said first antigen with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second antigen.

In another non-limiting embodiment, an antibody may be considered tobind a first antigen preferentially if it binds said first antigen withan off rate (k_(off)) that is less than the antibody's k_(off) for thesecond antigen. In another non-limiting embodiment, an antibody may beconsidered to bind a first antigen preferentially if it binds said firstantigen with an affinity that is at least one order of magnitude lessthan the antibody's k_(off) for the second antigen. In anothernon-limiting embodiment, an antibody may be considered to bind a firstantigen preferentially if it binds said first antigen with an affinitythat is at least two orders of magnitude less than the antibody'sk_(off) for the second antigen.

Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁴ M.More preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M,10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even more preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In specific embodiments, antibodies of the invention bind G-proteinChemokine Receptor (CCR5) polypeptides or fragments or variants thereofwith an off rate (k_(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻²sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. More preferably, antibodies of theinvention bind G-protein Chemokine Receptor (CCR5) polypeptides orfragments or variants thereof with an off rate (k_(off)) less than orequal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

In other embodiments, antibodies of the invention bind G-proteinChemokine Receptor (CCR5) polypeptides or fragments or variants thereofwith an on rate (k_(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹,5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably,antibodies of the invention bind G-protein Chemokine Receptor (CCR5)polypeptides or fragments or variants thereof with an on rate (k_(on))greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹,or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

In one embodiment of the present invention, antibodies thatimmunospecifically bind to a G-protein Chemokine Receptor (CCR5) or afragment or variant thereof, comprise a polypeptide having the aminoacid sequence of any one of the heavy chains expressed by ananti-G-protein Chemokine Receptor (CCR5) antibody expressing cell lineof the invention and/or any one of the light chains expressed by ananti-G-protein Chemokine Receptor (CCR5) antibody expressing cell lineof the invention. In another embodiment of the present invention,antibodies that immunospecifically bind to a G-protein ChemokineReceptor (CCR5) or a fragment or variant thereof, comprise a polypeptidehaving the amino acid sequence of any one of the VH domains of a heavychain expressed by an anti-G-protein Chemokine Receptor (CCR5) antibodyexpressing cell line of the invention and/or any one of the VL domainsof a light chain expressed by an anti-G-protein Chemokine Receptor(CCR5) antibody expressing cell line of the invention. In preferredembodiments, antibodies of the present invention comprise the amino acidsequence of a VH domain and VL domain expressed by a singleanti-G-protein Chemokine Receptor (CCR5) antibody expressing cell lineof the invention. In alternative embodiments, antibodies of the presentinvention comprise the amino acid sequence of a VH domain and a VLdomain expressed by two different anti-G-protein Chemokine Receptor(CCR5) antibody expressing cell lines of the invention. Moleculescomprising, or alternatively consisting of, antibody fragments orvariants of the VH and/or VL domains expressed by an anti-G-proteinChemokine Receptor (CCR5) antibody expressing cell line of the inventionthat immunospecifically bind to a G-protein Chemokine Receptor (CCR5)are also encompassed by the invention, as are nucleic acid moleculesencoding these VH and VL domains, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificallybind to a polypeptide, or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, a polypeptide having an amino acid sequence ofany one, two, three, or more of the VH CDRs contained in a heavy chainexpressed by one or more anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell lines of the invention. In particular, theinvention provides antibodies that immunospecifically bind a G-proteinChemokine Receptor (CCR5), comprising, or alternatively consisting of, apolypeptide having the amino acid sequence of a VH CDR1 contained in aheavy chain expressed by one or more anti-G-protein Chemokine Receptor(CCR5) antibody expressing cell lines of the invention. In anotherembodiment, antibodies that immunospecifically bind a G-proteinChemokine Receptor (CCR5), comprise, or alternatively consist of, apolypeptide having the amino acid sequence of a VH CDR2 contained in aheavy chain expressed by one or more anti-G-protein Chemokine Receptor(CCR5) antibody expressing cell lines of the invention. In a preferredembodiment, antibodies that immunospecifically bind a G-proteinChemokine Receptor (CCR5), comprise, or alternatively consist of apolypeptide having the amino acid sequence of a VH CDR3 contained in aheavy chain expressed by one or more anti-G-protein Chemokine Receptor(CCR5) antibody expressing cell lines of the invention. Moleculescomprising, or alternatively consisting of, these antibodies, orantibody fragments or variants thereof, that immunospecifically bind toG-protein Chemokine Receptor (CCR5) or a G-protein Chemokine Receptor(CCR5) fragment or variant thereof are also encompassed by theinvention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificallybind to a polypeptide, or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, a polypeptide having an amino acid sequence ofany one, two, three, or more of the VL CDRs contained in a light chainexpressed by one or more anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell lines of the invention. In particular, theinvention provides antibodies that immunospecifically bind a G-proteinChemokine Receptor (CCR5), comprising, or alternatively consisting of, apolypeptide having the amino acid sequence of a VL CDR1 contained in alight expressed by one or more anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell lines of the invention. In another embodiment,antibodies that immunospecifically bind a G-protein Chemokine Receptor(CCR5), comprise, or alternatively consist of, a polypeptide having theamino acid sequence of a VL CDR2 contained in a light chain expressed byone or more anti-G-protein Chemokine Receptor (CCR5) antibody expressingcell lines of the invention. In a preferred embodiment, antibodies thatimmunospecifically bind a G-protein Chemokine Receptor (CCR5), comprise,or alternatively consist of a polypeptide having the amino acid sequenceof a VL CDR3 contained in a light chain expressed by one or moreanti-G-protein Chemokine Receptor (CCR5) antibody expressing cell linesof the invention. Molecules comprising, or alternatively consisting of,these antibodies, or antibody fragments or variants thereof, thatimmunospecifically bind to G-protein Chemokine Receptor (CCR5) or aG-protein Chemokine Receptor (CCR5) fragment or variant thereof are alsoencompassed by the invention, as are nucleic acid molecules encodingthese antibodies, molecules, fragments and/or variants.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that immunospecifically bind to a G-protein Chemokine Receptor(CCR5) polypeptide or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, one, two, three, or more VH CDRs and one, two,three or more VL CDRs, as contained in a heavy chain or light chainexpressed by one or more anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell lines of the invention. In particular, theinvention provides for antibodies that immunospecifically bind to apolypeptide or polypeptide fragment or variant of a G-protein ChemokineReceptor (CCR5), wherein said antibodies comprise, or alternativelyconsist of, a VH CDR1 and a VL CDR1, a VH CDR1 and a VL CDR2, a VH CDR1and a VL CDR3, a VH CDR2 and a VL CDR1, VH CDR2 and VL CDR2, a VH CDR2and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VHCDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VLCDRs contained in a light chain or light chain expressed by one or moreanti-G-protein Chemokine Receptor (CCR5) antibody expressing cell linesof the invention. In a preferred embodiment, one or more of thesecombinations are from a single anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell line of the invention. Molecules comprising, oralternatively consisting of, fragments or variants of these antibodies,that immunospecifically bind to G-protein Chemokine Receptor (CCR5) arealso encompassed by the invention, as are nucleic acid moleculesencoding these antibodies, molecules, fragments or variants.

The present invention also provides for nucleic acid molecules,generally isolated, encoding an antibody of the invention (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof). In a specific embodiment, a nucleic acid moleculeof the invention encodes an antibody (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),comprising, or alternatively consisting of, a VH domain having an aminoacid sequence of any one of the VH domains of a heavy chain expressed byan anti-G-protein Chemokine Receptor (CCR5) antibody expressing cellline of the invention and a VL domain having an amino acid sequence of alight chain expressed by an anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell line of the invention. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, a VHdomain having an amino acid sequence of any one of the VH domains of aheavy chain expressed by an anti-G-protein Chemokine Receptor (CCR5)antibody expressing cell line of the invention or a VL domain having anamino acid sequence of a light chain expressed by an anti-G-proteinChemokine Receptor (CCR5) antibody expressing cell line of theinvention.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the VH domains and/or VL domains) describedherein, which antibodies immunospecifically bind to a G-proteinChemokine Receptor (CCR5) or fragment or variant thereof. Standardtechniques known to those of skill in the art can be used to introducemutations in the nucleotide sequence encoding a molecule of theinvention, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain,VLCDR1, VLCDR2, or VLCDR3. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind a G-protein Chemokine Receptor).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind a G-proteinChemokine Receptor) can be determined using techniques described hereinor by routinely modifying techniques known in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that immunospecifically binds G-proteinChemokine Receptor (CCR5) polypeptides or fragments or variants thereof,comprises, or alternatively consists of, an amino acid sequence encodedby a nucleotide sequence that hybridizes to a nucleotide sequence thatis complementary to that encoding one of the VH or VL domains expressedby one or more anti-G-protein Chemokine Receptor (CCR5) antibodyexpressing cell lines of the invention, under stringent conditions,e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C. followed by one or more washes in0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions,e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45°C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.,or under other stringent hybridization conditions which are known tothose of skill in the art (see, for example, Ausubel, F. M. et al.,eds., 1989, Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York atpages 6.3.1-6.3.6 and 2.10.3). Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a G-protein Chemokine Receptor (CCR5)polypeptide or fragments or variants of a G-protein Chemokine Receptor(CCR5) polypeptide, comprises, or alternatively consists of, a VH domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VHdomain of a heavy chain expressed by an anti-G-protein ChemokineReceptor (CCR5) antibody expressing cell line of the invention.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a G-protein Chemokine Receptor (CCR5)polypeptide or fragments or variants of a G-protein Chemokine Receptor(CCR5) polypeptide, comprises, or alternatively consists of, a VL domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VLdomain of a light chain expressed by an anti-G-protein ChemokineReceptor (CCR5) antibody expressing cell line of the invention.

The invention also encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that have one or more of the same biologicalcharacteristics as one or more of the antibodies described herein. By“biological characteristics” is meant, the in vitro or in vivoactivities or properties of the antibodies, such as, for example, theability to bind to G-protein Chemokine Receptor (CCR5) (e.g., G-proteinChemokine Receptor (CCR5) expressed on a cell surface, membrane-embeddedG-protein Chemokine Receptor (CCR5), and/or a fragment or variant ofG-protein Chemokine Receptor (CCR5)); the ability to substantiallyinhibit or abolish the binding of the G-protein Chemokine Receptor(CCR5) to a G-protein Chemokine Receptor (CCR5) ligand (e.g. MIP1-beta,see, e.g., Example 61); the ability to downregulate G-protein ChemokineReceptor (CCR5) expression on the surface of cells; the ability toinhibit or abolish G-protein Chemokine Receptor (CCR5) mediatedbiological activity (e.g., HIV binding to, infection (entryinto/fusion), and/or replication in, G-protein Chemokine Receptor (CCR5)expressing cells (see, e.g., Example 60), the ability to inhibit orabolish MIP1-beta induced chemotaxis of peripheral blood mononuclearcells PBMC (or other G-protein Chemokine Receptor (CCR5) expressingcells), or the ability to induce an intracellular calcium flux inG-protein Chemokine Receptor (CCR5) expressing cells, (see, e.g.,Example 63). Optionally, the antibodies of the invention will bind tothe same epitope as at least one of the antibodies specifically referredto herein. Such epitope binding can be routinely determined using assaysknown in the art.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that neutralize G-protein Chemokine Receptor (CCR5),said antibodies comprising, or alternatively consisting of, a portion(e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3) of aVH or VL domain of an antibody of the invention. An antibody that“neutralizes G-protein Chemokine Receptor (CCR5) or a fragment orvariant thereof” is, for example, an antibody that diminishes orabolishes the ability of G-protein Chemokine Receptor (CCR5) or afragment or variant thereof to bind to its ligand (e.g., HIV andMIP1-beta); that diminishes or abolishes MIP1-beta induced chemotaxis ofPBMC or other CCR5 expressing cell; and/or that abolishes or inhibitsthe G-protein Chemokine Receptor (CCR5) signaling cascade (e.g., calciumflux initiated by an activated G-protein Chemokine Receptor (CCR5), see,e.g., Example 63). In one embodiment, an antibody that neutralizesG-protein Chemokine Receptor (CCR5), comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainof an antibody of the invention, or a fragment or variant thereof and aVL domain of an antibody of the invention, or a fragment or variantthereof. In another embodiment, an antibody that neutralizes G-proteinChemokine Receptor (CCR5), comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain and a VLdomain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that neutralizes G-protein Chemokine Receptor (CCR5),comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain of an antibody of the invention, or afragment or variant thereof. In another embodiment, an antibody thatneutralizes G-protein Chemokine Receptor (CCR5), comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL domain of an antibody of the invention, or a fragment or variantthereof. In another embodiment, an antibody that neutralizes G-proteinChemokine Receptor (CCR5) or a fragment or variant thereof, comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a VH CDR domain of an antibody of the invention, or afragment or variant thereof. In a preferred embodiment, an antibody thatneutralizes G-protein Chemokine Receptor (CCR5) or a fragment or variantthereof, comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VH CDR3 of an antibody of the invention, ora fragment or variant thereof. In another embodiment, an antibody thatneutralizes G-protein Chemokine Receptor (CCR5) or a fragment or variantthereof, comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VL CDR of an antibody of the invention, ora fragment or variant thereof. In another preferred embodiment, anantibody that neutralizes G-protein Chemokine Receptor (CCR5) or afragment or variant thereof, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VL CDR3 of an antibodyof the invention, or a fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that reduces or abolishes the ability of HIV viruses,particularly those that utilize G-protein Chemokine Receptor (CCR5) as aco-receptor, to bind to, infect (enter into/fuse with), and/or replicatein G-protein Chemokine Receptor (CCR5) expressing cells, as determinedby any method known in the art such as, for example, the assaysdescribed Example 60. Said antibodies may comprise, or alternativelyconsist of, a portion (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, or VL CDR3) of a VH or VL domain having an amino acid sequence ofan antibody of the invention or a fragment or variant thereof. In oneembodiment, an antibody that reduces or abolishes the ability of HIVviruses, particularly those that utilize G-protein Chemokine Receptor(CCR5) as a co-receptor, to bind to, infect (enter into/fuse with),and/or replicate in G-protein Chemokine Receptor (CCR5) expressingcells, comprises, or alternatively consists of, a polypeptide having theamino acid sequence of a VH domain of an antibody of the invention, or afragment or variant thereof and a VL domain of an antibody of theinvention, or a fragment or variant thereof. In another embodiment, anantibody that reduces or abolishes the ability of HIV viruses,particularly those that utilize G-protein Chemokine Receptor (CCR5) as aco-receptor, to bind to, infect (enter into/fuse with), and/or replicatein G-protein Chemokine Receptor (CCR5) expressing cells, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH domain and a VL domain from a single antibody (or scFv or Fabfragment) of the invention, or fragments or variants thereof. In oneembodiment, an antibody that reduces or abolishes the ability of HIVviruses, particularly those that utilize G-protein Chemokine Receptor(CCR5) as a co-receptor, to bind to, infect (enter into/fuse with),and/or replicate in G-protein Chemokine Receptor (CCR5) expressingcells, comprises, or alternatively consists of, a polypeptide having theamino acid sequence of a VH domain of an antibody of the invention, or afragment or variant thereof. In another embodiment, an antibody thatreduces or abolishes the ability of HIV viruses, particularly those thatutilize G-protein Chemokine Receptor (CCR5) as a co-receptor, to bindto, infect (enter into/fuse with), and/or replicate in G-proteinChemokine Receptor (CCR5) expressing cells, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VL domainof an antibody of the invention, or a fragment or variant thereof. In apreferred embodiment, an antibody that reduces or abolishes the abilityof HIV viruses, particularly those that utilize G-protein ChemokineReceptor (CCR5) as a co-receptor, to bind to, infect (enter into/fusewith), and/or replicate in G-protein Chemokine Receptor (CCR5)expressing cells, comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VH CDR3 of an antibody of theinvention, or a fragment or variant thereof. In another preferredembodiment, an antibody that reduces or abolishes the ability of HIVviruses, particularly those that utilize G-protein Chemokine Receptor(CCR5) as a co-receptor, to bind to, infect (enter into/fuse with),and/or replicate in G-protein Chemokine Receptor (CCR5) expressingcells, comprises, or alternatively consists of, a polypeptide having theamino acid sequence of a VL CDR3 of an antibody of the invention, or afragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibits or abolishes MIP1-beta inducedchemotaxis of peripheral blood mononuclear cells PBMC or other G-proteinChemokine Receptor (CCR5) expressing cells, as determined by any methodknown in the art such as, for example, the assays described in Example62. Said antibodies may comprise, or alternatively consist of, a portion(e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of a VHor VL domain having an amino acid sequence of an antibody of theinvention or a fragment or variant thereof. In one embodiment, anantibody that inhibits or abolishes MIP1-beta induced chemotaxis ofperipheral blood mononuclear cells PBMC or other G-protein ChemokineReceptor (CCR5) expressing cells, comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH domain of anantibody of the invention, or a fragment or variant thereof and a VLdomain of an antibody of the invention, or a fragment or variantthereof. In another embodiment, an antibody that inhibits or abolishesMIP1-beta induced chemotaxis of peripheral blood mononuclear cells PBMCor other G-protein Chemokine Receptor (CCR5) expressing cells,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain and a VL domain from a single antibody (orscFv or Fab fragment) of the invention, or fragments or variantsthereof. In one embodiment, an antibody that inhibits or abolishesMIP1-beta induced chemotaxis of peripheral blood mononuclear cells PBMCor other G-protein Chemokine Receptor (CCR5) expressing cells,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain of an antibody of the invention, or afragment or variant thereof. In another embodiment, an antibody thatinhibits or abolishes MIP1-beta induced chemotaxis of peripheral bloodmononuclear cells PBMC or other G-protein Chemokine Receptor (CCR5)expressing cells, comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VL domain of an antibody of theinvention, or a fragment or variant thereof. In a preferred embodiment,an antibody that inhibits or abolishes MIP1-beta induced chemotaxis ofperipheral blood mononuclear cells PBMC or other G-protein ChemokineReceptor (CCR5) expressing cells, comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH CDR3 of anantibody of the invention, or a fragment or variant thereof. In anotherpreferred embodiment, an antibody that inhibits or abolishes MIP1-betainduced chemotaxis of peripheral blood mononuclear cells PBMC or otherG-protein Chemokine Receptor (CCR5) expressing cells, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL CDR3 of an antibody of the invention, or a fragment or variantthereof. Nucleic acid molecules encoding these antibodies are alsoencompassed by the invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that downregulates the cell-surface expression ofG-protein Chemokine Receptor (CCR5), as determined by any method knownin the art such as, for example, FACS analysis/the assays described inExamples 61 or 63. By way of a non-limiting hypothesis, such downregulation may be the result of antibody induced internalization ofG-protein Chemokine Receptor (CCR5). Said antibodies may comprise, oralternatively consist of, a portion (e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, or VL CDR3) of a VH or VL domain having an amino acidsequence of an antibody of the invention or a fragment or variantthereof. In one embodiment, an antibody that downregulates thecell-surface expression of G-protein Chemokine Receptor (CCR5),comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain of an antibody of the invention, or afragment or variant thereof and a VL domain of an antibody of theinvention, or a fragment or variant thereof. In another embodiment, anantibody that downregulates the cell-surface expression of G-proteinChemokine Receptor (CCR5), comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain and a VLdomain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that downregulates the cell-surface expression of G-proteinChemokine Receptor (CCR5), comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain of an antibodyof the invention, or a fragment or variant thereof. In anotherembodiment, an antibody that downregulates the cell-surface expressionof G-protein Chemokine Receptor (CCR5), comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VL domainof an antibody of the invention, or a fragment or variant thereof. In apreferred embodiment, an antibody that downregulate the cell-surfaceexpression of G-protein Chemokine Receptor (CCR5), comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH CDR3 of an antibody of the invention, or a fragment or variantthereof. In another preferred embodiment, an antibody that downregulatesthe cell-surface expression of G-protein Chemokine Receptor (CCR5),comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VL CDR3 of an antibody of the invention, or afragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that enhance the activity of G-protein ChemokineReceptor (CCR5), said antibodies comprising, or alternatively consistingof, a portion (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VLCDR3) of a VH or VL domain of an antibody of the invention, or afragment or variant thereof. By way of non-limiting example, an antibodythat “enhances the activity of G-protein Chemokine Receptor (CCR5) or afragment or variant thereof” is an antibody increases the ability ofG-protein Chemokine Receptor (CCR5) to bind to stimulate chemotaxis ofPBMC (or other G-protein Chemokine Receptor (CCR5) expressing cells),and/or to stimulate the G-protein Chemokine Receptor (CCR5) signalingcascade (e.g., to initiate an intracellular calcium flux, See Example63). In one embodiment, an antibody that that enhances the activity ofG-protein Chemokine Receptor (CCR5), comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainof an antibody of the invention, or a fragment or variant thereof and aVL domain of an antibody of the invention, or a fragment or variantthereof. In another embodiment, an antibody that enhances the activityof G-protein Chemokine Receptor (CCR5), comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainand a VL domain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that enhances the activity of G-protein Chemokine Receptor(CCR5) or a fragment or variant thereof, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainof an antibody of the invention, or a fragment or variant thereof. Inanother embodiment, an antibody that enhances the activity of G-proteinChemokine Receptor (CCR5) or a fragment or variant thereof, comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a VL domain of an antibody of the invention, or a fragmentor variant thereof. In another embodiment, an antibody that enhances theactivity of G-protein Chemokine Receptor (CCR5) or a fragment or variantthereof, comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VH CDR domain referred to in Table 2, or afragment or variant thereof. In a preferred embodiment, an antibody thatenhances the activity of G-protein Chemokine Receptor (CCR5) or afragment or variant thereof, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH CDR3 of an antibodyof the invention, or a fragment or variant thereof. In anotherembodiment, an antibody that enhances G-protein Chemokine Receptor(CCR5) or a fragment or variant thereof, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VL CDRdomain of an antibody of the invention, or a fragment or variantthereof. In another preferred embodiment, an antibody that enhances theactivity of G-protein Chemokine Receptor (CCR5) or a fragment or variantthereof, comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VL CDR3 of an antibody of the invention, ora fragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

The present invention also provides for fusion proteins comprising, oralternatively consisting of, an antibody (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that immunospecifically binds to G-protein ChemokineReceptor (CCR5), and a heterologous polypeptide. Preferably, theheterologous polypeptide to which the antibody is fused to is useful forfunction or is useful to target the G-protein Chemokine Receptor (CCR5)expressing cells, including but not limited to, MIP-1-beta; a CD4binding polypeptide such as an anti-CD4 antibody; a CXCR4 bindingpolypeptides such as stromal derived factor 1-alpha (SDF1-alpha); and/ora CCR3 binding protein, such as MIP1-alpha). In an alternative preferredembodiment, the heterologous polypeptide to which the antibody is fusedto is useful for T cell, macrophage, and/or monocyte cell function or isuseful to target the antibody to a T cell, macrophage, or monocyte,including but not limited to, MIP-1-beta; a CD4 binding polypeptide suchas an anti-CD4 antibody; a CXCR4 binding polypeptides such as stromalderived factor 1-alpha (SDF1-alpha); and/or a CCR3 binding protein, suchas MIP1-alpha). In one embodiment, a fusion protein of the inventioncomprises, or alternatively consists of, a polypeptide having the aminoacid sequence of any one or more of the VH domains of an antibody of theinvention or the amino acid sequence of any one or more of the VLdomains of an antibody of the invention or fragments or variantsthereof, and a heterologous polypeptide sequence. In another embodiment,a fusion protein of the present invention comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of any one,two, three, or more of the VH CDRs of an antibody of the invention, orthe amino acid sequence of any one, two, three, or more of the VL CDRsof an antibody of the invention, or fragments or variants thereof, and aheterologous polypeptide sequence. In a preferred embodiment, the fusionprotein comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of, a VH CDR3 of an antibody of the invention,or fragment or variant thereof, and a heterologous polypeptide sequence,which fusion protein immunospecifically binds to G-protein ChemokineReceptor (CCR5). In another embodiment, a fusion protein comprises, oralternatively consists of a polypeptide having the amino acid sequenceof at least one VH domain of an antibody of the invention and the aminoacid sequence of at least one VL domain of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. Preferably, the VH and VL domains of the fusion proteincorrespond to a single antibody (or scFv or Fab fragment) of theinvention. In yet another embodiment, a fusion protein of the inventioncomprises, or alternatively consists of a polypeptide having the aminoacid sequence of any one, two, three or more of the VH CDRs of anantibody of the invention and the amino acid sequence of any one, two,three or more of the VL CDRs of an antibody of the invention, orfragments or variants thereof, and a heterologous polypeptide sequence.Preferably, two, three, four, five, six, or more of the VHCDR(s) orVLCDR(s) correspond to single antibody (or scFv or Fab fragment) of theinvention. Nucleic acid molecules encoding these fusion proteins arealso encompassed by the invention.

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

By way of another non-limiting example, antibodies of the invention maybe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention may be used forepitope mapping to identify the epitope(s) bound by the antibody.Epitopes identified in this way may, in turn, for example, be used asvaccine candidates, i.e., to immunize an individual to elicit antibodiesagainst the naturally occurring forms of G-protein Chemokine Receptor.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 or P3X63-AG8.653 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, NY, which is hereby incorporated in its entirety byreference herein. The source of B cells for transformation is commonlyhuman peripheral blood, but B cells for transformation may also bederived from other sources including, but not limited to, lymph nodes,tonsil, spleen, tumor tissue, and infected tissues. Tissues aregenerally made into single cell suspensions prior to EBV transformation.Additionally, steps may be taken to either physically remove orinactivate T cells (e.g., by treatment with cyclosporin A) in Bcell-containing samples, because T cells from individuals seropositivefor anti-EBV antibodies can suppress B cell immortalization by EBV. Ingeneral, the sample containing human B cells is inoculated with EBV, andcultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human x mouse; e.g., SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Fremont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby activate orblock its biological activity.

Intrabodies are antibodies, often scFvs, that expressed from arecombinant nucleic acid molecule and engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm). Intrabodies may be used, for example, to ablate thefunction of a protein to which the intrabody binds. The expression ofintrabodies may also be regulated through the use of inducible promotersin the nucleic acid expression vector comprising the intrabody.Intrabodies of the invention can be produced using methods known in theart, such as those disclosed and reviewed in Chen et al., Hum. GeneTher. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997);Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba etal., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz andSteipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods231:207-222 (1999); and references cited therein. In particular, a CCR5intrabody has been produced by Steinberger et al., Proc. Natl. Acad.Sci. USA 97:805-810 (2000).

XenoMouse Technology

Antibodies in accordance with the invention are preferably prepared bythe utilization of a transgenic mouse that has a substantial portion ofthe human antibody producing genome inserted but that is rendereddeficient in the production of endogenous, murine, antibodies (e.g.,XenoMouse strains available from Abgenix Inc., Fremont, Calif.). Suchmice, then, are capable of producing human immunoglobulin molecules andantibodies and are deficient in the production of murine immunoglobulinmolecules and antibodies. Technologies utilized for achieving the sameare disclosed in the patents, applications, and references disclosedherein.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs) an important milestone towards fulfilling the promiseof antibody therapy in human disease.

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized monoclonalantibodies and thus to increase the efficacy and safety of theadministered antibodies. The use of fully human antibodies can beexpected to provide a substantial advantage in the treatment of chronicand recurring human diseases, such as cancer, which require repeatedantibody administrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human Monoclonalantibodies with the desired specificity could be readily produced andselected.

This general strategy was demonstrated in connection with the generationof the first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineeredwith yeast artificial chromosomes (YACS) containing 245 kb and 10 190kb-sized germline configuration fragments of the human heavy chain locusand kappa light chain locus, respectively, which contained core variableand constant region sequences. Id. The human Ig containing YACs provedto be compatible with the mouse system for both rearrangement andexpression of antibodies and were capable of substituting for theinactivated mouse Ig genes. This was demonstrated by their ability toinduce B-cell development, to produce an adult-like human repertoire offully human antibodies, and to generate antigen-specific humanmonoclonal antibodies. These results also suggested that introduction oflarger portions of the human Ig loci containing greater numbers of Vgenes, additional regulatory elements, and human Ig constant regionsmight recapitulate substantially the full repertoire that ischaracteristic of the human humoral response to infection andimmunization. The work of Green et al. was recently extended to theintroduction of greater than approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse™ mice. See Mendezet al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J Exp.Med. 188:483-495 (1998), Green, Journal of Immunological Methods231:11-23 (1999) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996, the disclosures of which are hereby incorporated byreference.

Such approach is further discussed and delineated in U.S. patentapplication Ser. No. 07/466,008, filed Jan. 12, 1990, 07/710,515, filedNov. 8, 1990, 07/919,297, filed Jul. 24, 1992, 07/922,649, filed Jul.30, 1992, filed 08/031,801, filed Mar. 15, 1993, 08/112,848, filed Aug.27, 1993, 08/234,145, filed Apr. 28, 1994, 08/376,279, filed Jan. 20,1995, 08/430, 938, Apr. 27, 1995, 0-8/464,584, filed Jun. 5, 1995,08/464,582, filed Jun. 5, 1995, 08/471,191, filed Jun. 5, 1995,08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5, 1995,08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5, 1995,08/462,513, filed Jun. 5, 1995, 08/724,752, filed Oct. 2, 1996, and08/759,620, filed Dec. 3, 1996. See also Mendez et al. Nature Genetics15:146-156 (1997) and Green and Jakobovits J Exp. Med. 188:483 495(1998). See also European Patent No., EP 0 471151 B1, grant publishedJun. 12, 1996, International Patent Application No., WO 94/02602,published Feb. 3, 1994, International Patent Application No., WO96/34096, published Oct. 31, 1996, and WO 98/24893, published Jun. 11,1998. The disclosures of each of the above-cited patents, applications,and references are hereby incorporated by reference in their entirety.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against G-protein Chemokine Receptor (CCR5) polypeptides inorder to vitiate concerns and/or effects of HAMA or HACA responses.

Monoclonal antibodies specific for G-protein Chemokine Receptor (CCR5)polypeptides were prepared using hybridoma technology. (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 571-681(1981)). Briefly, XenoMouse™ mice were immunized with cells transfectedwith a G-protein Chemokine Receptor (CCR5) expression vector (fordetails, see Example 54). After immunization, the splenocytes of suchmice were extracted and fused with a suitable myeloma cell line. Anysuitable myeloma cell line may be employed in accordance with thepresent invention; however, it is preferable to employ the parentmyeloma cell line (P3X63-AG8.653), available from the ATCC. Afterfusion, the resulting hybridoma cells are selectively maintained in HATmedium, and then cloned by limiting dilution as described by Wands etal. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtainedthrough such a selection are then assayed to identify clones whichsecrete antibodies capable of binding the G-protein Chemokine Receptor(CCR5) polypeptides.

The present invention is directed to fully human antibodies, generallyisolated, that immunospecifically bind G-protein Chemokine Receptor(CCR5) polypeptides. Essentially, XenoMouse lines of mice from Abgenix,Inc. (Fremont, Calif.) expressing human antibodies were immunized withG-protein Chemokine Receptor (CCR5) expressing cells (for details ofimmunization protocols, see Example 54); spleen and/or lymph node cells(containing B-cells) were recovered from the mice that had high titersof anti-G-protein Chemokine Receptor (CCR5) antibodies; and suchrecovered cells were fused with a myeloid-type cell line to prepareimmortal hybridoma cell lines. Hybridoma cell lines were screened toselect and identify hybridoma cell lines that produced antibodiesspecific to the immunogen. We utilized these techniques in accordancewith the present invention for the preparation of antibodies specific toG-protein Chemokine Receptor (CCR5) polypeptides. Herein, we describethe production of multiple hybridoma cell lines that produce antibodiesspecific to G-protein Chemokine Receptor (CCR5) polypeptides. Further,we provide a characterization of the antibodies produced by such celllines.

The antibodies derived from hybridoma cell lines discussed herein arelisted in Table 2. Preferred antibodies of the invention include,antibodies expressed by the following cell lines: XF11.1D8, XF11.4D10,XF11.4C4, XF11.5H1, XF11.1G8, XF22.3C9.6, XF22.9E6, XF27/28.7D5,XF27/28.18B5, XF27/28.25G10, XF27/28.36A12, XF27/28.36F11, XF27/28.43E2,and XF27/28.55G4. XenoMouse strains of mice from Abgenix, Inc. expresshuman kappa light chains with either human IgG1, IgG2, or IgG4. Each ofthe XenoMouse strains can also produce antibodies of the human IgMisotype. The IgG2 expressing strain was used to make the cell lines andantibodies of the present invention, thus each of the antibodiesproduced by cell lines are either fully human IgG2 heavy chains withhuman kappa light chains or IgM heavy chains with human kappa lightchains. These hybridoma cell lines were deposited with the American TypeCulture Collection (“ATCC”) on the date listed in Table 2, and givenATCC Deposit Numbers listed in Table 2. The ATCC is located at 10801University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC depositwas made pursuant to the terms of the Budapest Treaty on theinternational recognition of the deposit of microorganisms for purposesof patent procedure.

Hybridoma XF11.1D8 was deposited at the ATCC on Feb. 7, 2001 and givenATCC Deposit Number PTA-3030. Hybridoma XF11.4D10 was deposited at theATCC on Feb. 7, 2001 and given ATCC Deposit Number PTA-3026. HybridomaXF11.4C4 was deposited at the ATCC on Feb. 7, 2001 and given ATCCDeposit Number PTA-3028. Hybridoma XF11.5H1 was deposited at the ATCC onFeb. 7, 2001 and given ATCC Deposit Number PTA-3029. Hybridoma XF11.1G8was deposited at the ATCC on Feb. 7, 2001 and given ATCC Deposit NumberPTA-3027. Hybridoma XF3.5F1 was deposited at the ATCC on Mar. 2, 2001and given ATCC Deposit Number PTA-3147. Hybridoma XF11.1F8 was depositedat the ATCC on Mar. 2, 2001 and given ATCC Deposit Number PTA-3150.Hybridoma XF3.6A2 was deposited at the ATCC on Mar. 2, 2001 and givenATCC Deposit Number PTA-3148. Hybridoma XF3.10B8 was deposited at theATCC on Mar. 2, 2001 and given ATCC Deposit Number PTA-3151. HybridomaXF22.3C9.6 was deposited at the ATCC on Sep. 12, 2001 and given ATCCDeposit Number PTA-3702. Hybridoma XF22.9E6 was deposited at the ATCC onNov. 14, 2001 and given ATCC Deposit Number PTA-3859. HybridomaXF27/28.7D5 was deposited at the ATCC on Feb. 1, 2002 and given ATCCDeposit Number PTA-4049. Hybridoma XF27/28.18B5 was deposited at theATCC on Feb. 1, 2002 and given ATCC Deposit Number PTA-4050. HybridomaXF27/28.25G10 was deposited at the ATCC on Feb. 1, 2002 and given ATCCDeposit Number PTA-4051. Hybridoma XF27/28.36A12 was deposited at theATCC on Feb. 1, 2002 and given ATCC Deposit Number PTA-4052. HybridomaXF27/28.36F11 was deposited at the ATCC on Feb. 1, 2002 and given ATCCDeposit Number PTA-4053. Hybridoma XF27/28.43E2 was deposited at theATCC on Feb. 1, 2002 and given ATCC Deposit Number PTA-4054. The ATCCDeposit Numbers and the Hybridoma designations are also presented inTable 2.

TABLE 2 Hybridoma Cell Lines Expressing anti-G-protein ChemokineReceptor (CCR5) Receptor Antibodies ATCC Deposit Hybridoma ATCC DepositNumber Date XF11.1D8 PTA-3030 Feb. 7, 2001 XF11.4D10 PTA-3026 Feb. 7,2001 XF11.4C4 PTA-3028 Feb. 7, 2001 XF11.5H1 PTA-3029 Feb. 7, 2001XF11.1G8 PTA-3027 Feb. 7, 2001 XF3.5F1 PTA-3147 Mar. 2, 2001 XF11.1F8PTA-3150 Mar. 2, 2001 XF3.6A2 PTA-3148 Mar. 2, 2001 XF3.10B8 PTA-3151Mar. 2, 2001 XF22.3C9.6 PTA-3702 Sep. 12, 2001 XF22.9E6 PTA-3859 Nov.14, 2001 XF27/28.7D5 PTA-4049 Feb. 1, 2002 XF27/28.18B5 PTA-4050 Feb. 1,2002 XF27/28.25G10 PTA-4051 Feb. 1, 2002 XF27/28.36A12 PTA-4052 Feb. 1,2002 XF27/28.36F11 PTA-4053 Feb. 1, 2002 XF27/28.43E2 PTA-4054 Feb. 1,2002 XF27/28.55G4

In one embodiment, the present invention provides hybridoma cell linesexpressing an antibody of the invention. In specific embodiments, thehybridoma cell line of the invention is XF11.1D8. In another specificembodiment, the hybridoma cell line of the invention is XF11.4D10. Inanother specific embodiment, the hybridoma cell line of the invention isXF11.4C4. In another specific embodiment, the hybridoma cell line of theinvention is XF11.5H1. In another specific embodiment, the hybridomacell line of the invention is XF11.1G8. In another specific embodiment,the hybridoma cell line of the invention is XF3.5F1. In another specificembodiment, the hybridoma cell line of the invention is XF11.1F8. Inanother specific embodiment, the hybridoma cell line of the invention isXF3.6A2. In another specific embodiment, the hybridoma cell line of theinvention is XF3.10B8. In another specific embodiment, the hybridomacell line of the invention is XF22.3C9.6. In another specificembodiment, the hybridoma cell line of the invention is XF22.9E6. Inanother specific embodiment, the hybridoma cell line of the invention isXF27/28.7D5. In another specific embodiment, the hybridoma cell line ofthe invention is XF27/28.18B5. In another specific embodiment, thehybridoma cell line of the invention is XF27/28.25G10. In anotherspecific embodiment, the hybridoma cell line of the invention isXF27/28.36A12. In another specific embodiment, the hybridoma cell lineof the invention is XF27/28.36F11. In another specific embodiment, thehybridoma cell line of the invention is XF27/28.43E2. In anotherspecific embodiment, the hybridoma cell line of the invention isXF27/28.55G4.

The present invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that immunospecifically bind to a G-protein ChemokineReceptor (CCR5) polypeptide or a fragment, variant, or fusion proteinthereof. A G-protein Chemokine Receptor (CCR5) polypeptide includes, butis not limited to, the G-protein Chemokine Receptor (CCR5) polypeptideof SEQ ID NO:2 or the polypeptide encoded by the DNA in clone HDGNR10contained in ATCC Deposit 97183 deposited Jun. 1, 1995; G-proteinChemokine Receptor (CCR5) may be produced through recombinant expressionof nucleic acids encoding the polypeptides of SEQ ID NOS:2 or theHDGNR10 DNA in ATCC Deposit Number 97183).

In one embodiment of the present invention, antibodies thatimmunospecifically bind to a G-protein Chemokine Receptor (CCR5) or afragment or variant thereof, comprise a polypeptide having the aminoacid sequence of any one of the heavy chains expressed by at least oneof the cell lines referred to in Table 2 and/or any one of the lightchains expressed by at least one of the cell lines referred to in Table2. In another embodiment of the present invention, antibodies thatimmunospecifically bind to a G-protein Chemokine Receptor (CCR5) or afragment or variant thereof, comprise a polypeptide having the aminoacid sequence of any one of the VH domains of a heavy chain expressed byat least one of the cell lines referred to in Table 2 and/or any one ofthe VL domains of a light chain expressed by at least one of the celllines referred to in Table 2. In preferred embodiments, antibodies ofthe present invention comprise the amino acid sequence of a VH domainand VL domain expressed by the same cell line selected from the groupconsisting of cell lines referred to in Table 2. In alternativeembodiments, antibodies of the present invention comprise the amino acidsequence of a VH domain and a VL domain from different cell linesreferred to in Table 2. Molecules comprising, or alternativelyconsisting of, antibody fragments or variants of the VH and/or VLdomains expressed by at least one of the cell lines referred to in Table2 that immunospecifically bind to a G-protein Chemokine Receptor (CCR5)are also encompassed by the invention, as are nucleic acid moleculesencoding these VH and VL domains, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificallybind to a polypeptide, or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, a polypeptide having an amino acid sequence ofany one, two, three, or more of the VH CDRs contained in a heavy chainexpressed by one or more cell lines referred to in Table 2. Inparticular, the invention provides antibodies that immunospecificallybind a G-protein Chemokine Receptor (CCR5), comprising, or alternativelyconsisting of, a polypeptide having the amino acid sequence of a VH CDR1contained in a heavy chain expressed by one or more cell lines referredto in Table 2. In another embodiment, antibodies that immunospecificallybind a G-protein Chemokine Receptor (CCR5), comprise, or alternativelyconsist of, a polypeptide having the amino acid sequence of a VH CDR2contained in a heavy chain expressed by one or more cell lines referredto in Table 2. In a preferred embodiment, antibodies thatimmunospecifically bind a G-protein Chemokine Receptor (CCR5), comprise,or alternatively consist of a polypeptide having the amino acid sequenceof a VH CDR3 contained in a heavy chain expressed by one or more celllines referred to in Table 2. Molecules comprising, or alternativelyconsisting of, these antibodies, or antibody fragments or variantsthereof, that immunospecifically bind to G-protein Chemokine Receptor(CCR5) or a G-protein Chemokine Receptor (CCR5) fragment or variantthereof are also encompassed by the invention, as are nucleic acidmolecules encoding these antibodies, molecules, fragments and/orvariants.

The present invention also provides antibodies that immunospecificallybind to a polypeptide, or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, a polypeptide having an amino acid sequence ofany one, two, three, or more of the VL CDRs contained in a light chainexpressed by one or more cell lines referred to in Table 2. Inparticular, the invention provides antibodies that immunospecificallybind a G-protein Chemokine Receptor (CCR5), comprising, or alternativelyconsisting of, a polypeptide having the amino acid sequence of a VL CDR1contained in a light chain expressed by one or more cell lines referredto in Table 2. In another embodiment, antibodies that immunospecificallybind a G-protein Chemokine Receptor (CCR5), comprise, or alternativelyconsist of, a polypeptide having the amino acid sequence of a VL CDR2contained in a light chain expressed by one or more cell lines referredto in Table 2. In a preferred embodiment, antibodies thatimmunospecifically bind a G-protein Chemokine Receptor (CCR5), comprise,or alternatively consist of a polypeptide having the amino acid sequenceof a VL CDR3 contained in a light chain expressed by one or more celllines referred to in Table 2. Molecules comprising, or alternativelyconsisting of, these antibodies, or antibody fragments or variantsthereof, that immunospecifically bind to G-protein Chemokine Receptor(CCR5) or a G-protein Chemokine Receptor (CCR5) fragment or variantthereof are also encompassed by the invention, as are nucleic acidmolecules encoding these antibodies, molecules, fragments and/orvariants.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that immunospecifically bind to a G-protein Chemokine Receptor(CCR5) polypeptide or polypeptide fragment or variant of a G-proteinChemokine Receptor (CCR5), wherein said antibodies comprise, oralternatively consist of, one, two, three, or more VH CDRs and one, two,three or more VL CDRs, as contained in a heavy chain or light chainexpressed by one or more cell lines referred to in Table 2. Inparticular, the invention provides for antibodies thatimmunospecifically bind to a polypeptide or polypeptide fragment orvariant of a G-protein Chemokine Receptor (CCR5), wherein saidantibodies comprise, or alternatively consist of, a VH CDR1 and a VLCDR1, a VH CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and aVL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and aVH CDR 1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or anycombination thereof, of the VH CDRs and VL CDRs contained in a heavychain or light chain expressed by one or more cell lines referred to inTable 2. In a preferred embodiment, one or more of these combinationsare from the same scFv as disclosed in Table 2. Molecules comprising, oralternatively consisting of, fragments or variants of these antibodies,that immunospecifically bind to G-protein Chemokine Receptor (CCR5) arealso encompassed by the invention, as are nucleic acid moleculesencoding these antibodies, molecules, fragments or variants.

Nucleic Acid Molecules Encoding Anti-G-protein Chemokine Receptor (CCR5)Antibodies Corresponding to Antibodies Derived from Xenomouse Strains.

The present invention also provides for nucleic acid molecules,generally isolated, encoding an antibody of the invention (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof). In a specific embodiment, a nucleic acid moleculeof the invention encodes an antibody (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),comprising, or alternatively consisting of, a VH domain having an aminoacid sequence of any one of the VH domains of a heavy chain expressed byat least one of the cell lines referred to in Table 2 and a VL domainhaving an amino acid sequence of a light chain expressed by at least oneof the cell lines referred to in Table 2. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, a VHdomain having an amino acid sequence of any one of the VH domains of aheavy chain expressed by at least one of the cell lines referred to inTable 2 or a VL domain having an amino acid sequence of a light chainexpressed by at least one of the cell lines referred to in Table 2.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the VH domains and/or VL domains) describedherein, which antibodies immunospecifically bind to a G-proteinChemokine Receptor (CCR5) or fragment or variant thereof. Standardtechniques known to those of skill in the art can be used to introducemutations in the nucleotide sequence encoding a molecule of theinvention, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain,VLCDR1, VLCDR2, or VLCDR3. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind a G-protein Chemokine Receptor).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind a G-proteinChemokine Receptor) can be determined using techniques described hereinor by routinely modifying techniques known in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that immunospecifically binds G-proteinChemokine Receptor (CCR5) polypeptides or fragments or variants thereof,comprises, or alternatively consists of, an amino acid sequence encodedby a nucleotide sequence that hybridizes to a nucleotide sequence thatis complementary to that encoding one of the VH or VL domains expressedby one or more cell lines referred to in Table 2 under stringentconditions, e.g., hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC atabout 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about68° C., or under other stringent hybridization conditions which areknown to those of skill in the art (see, for example, Ausubel, F. M. etal., eds., 1989, Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York atpages 6.3.1-6.3.6 and 2.10.3). Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a G-protein Chemokine Receptor (CCR5)polypeptide or fragments or variants of a G-protein Chemokine Receptor(CCR5) polypeptide, comprises, or alternatively consists of, a VH domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VHdomain of a heavy chain expressed by at least one of the cell linesreferred to in Table 2.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a G-protein Chemokine Receptor (CCR5)polypeptide or fragments or variants of a G-protein Chemokine Receptor(CCR5) polypeptide, comprises, or alternatively consists of, a VL domainhaving an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to the amino acid sequence of a VLdomain of a light chain expressed by at least one of the cell linesreferred to in Table 2.

Polynucleotides Encoding Antibodies

Antibodies of the invention (including antibody fragments or variants)can be produced by any method known in the art. For example, it will beappreciated that antibodies in accordance with the present invention canbe expressed in cell lines other than hybridoma cell lines. Sequencesencoding the cDNAs or genomic clones for the particular antibodies canbe used for transformation of a suitable mammalian or nonmammalian hostcells or to generate phage display libraries, for example. Additionally,polypeptide antibodies of the invention may be chemically synthesized orproduced through the use of recombinant expression systems.

One way to produce the antibodies of the invention would be to clone theVH and/or VL domains expressed by any one or more of the hybridoma celllines referred to in Table 2. In order to isolate the VH and VL domainsfrom the hybridoma cell lines, PCR primers including VH or VL nucleotidesequences (See Example 55), may be used to amplify the expressed VH andVL sequences contained in total RNA isolated from hybridoma cell lines.The PCR products may then be cloned using vectors, for example, whichhave a PCR product cloning site consisting of a 5′ and 3′ single Tnucleotide overhang, that is complementary to the overhanging singleadenine nucleotide added onto the 5′ and 3′ end of PCR products by manyDNA polymerases used for PCR reactions. The VH and VL domains can thenbe sequenced using conventional methods known in the art.

The cloned VH and VL genes may be placed into one or more suitableexpression vectors. By way of non-limiting example, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site may be used to amplifythe VH or VL sequences. Utilizing cloning techniques known to those ofskill in the art, the PCR amplified VH domains may be cloned intovectors expressing the appropriate immunoglobulin constant region, e.g.,the human IgG1 or IgG4 constant region for VH domains, and the humankappa or lambda constant regions for kappa and lambda VL domains,respectively. Preferably, the vectors for expressing the VH or VLdomains comprise a promoter suitable to direct expression of the heavyand light chains in the chosen expression system, a secretion signal, acloning site for the immunoglobulin variable domain, immunoglobulinconstant domains, and a selection marker such as neomycin. The VH and VLdomains may also be cloned into a single vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art (See, forexample, Guo et al., J. Clin. Endocrinol. Metab. 82:925-31 (1997), andAmes et al., J. Immunol. Methods 184:177-86 (1995) which are hereinincorporated in their entireties by reference).

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2 or a polypeptide encoded by the deposited clone.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence(See Example 55) or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it may be useful to express the VH and VL domains ofthe heavy and light chains of one or more antibodies of the invention assingle chain antibodies or Fab fragments in a phage display library. Forexample, the cDNAs encoding the VH and VL domains of one or moreantibodies of the invention may be expressed in all possiblecombinations using a phage display library, allowing for the selectionof VH/VL combinations that bind a G-protein Chemokine Receptor (CCR5)polypeptides with preferred binding characteristics such as improvedaffinity or improved off rates. Additionally, VH and VL segments—the CDRregions of the VH and VL domains of one or more antibodies of theinvention, in particular, may be mutated in vitro. Expression of VH andVL domains with “mutant” CDRs in a phage display library allows for theselection of VH/VL combinations that bind a G-protein Chemokine Receptor(CCR5) receptor polypeptides with preferred binding characteristics suchas improved affinity or improved off rates.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. The DNAencoding the VH and VL domains are joined together by an scFv linker byPCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13 and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to an antigen ofinterest (i.e., a G-protein Chemokine Receptor polypeptide or a fragmentthereof) can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies of the present invention include, but are not limited to,those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18719; WO 93/11236; WO95/15982; WO 95/20401; WO 97/13844; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,717; 5,780,225; 5,658,727; 5,735,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis, by intracellular immunization (i.e., intrabody technology),or preferably, by recombinant expression techniques. Methods ofproducing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availability of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. Vectors that use glutamine synthase as the selectablemarker include, but are not limited to, the pEE6 expression vectordescribed in Stephens and Cockett, Nucl. Acids. Res 17:7110 (1989). Aglutamine synthase expression system and components thereof are detailedin PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are incorporated in their entireties by referenceherein. Additionally, glutamine synthase expression vectors that may beused according to the present invention are commercially available fromsuppliers, including, for example Lonza Biologics, Inc. (Portsmouth,N.H.). Expression and production of monoclonal antibodies using a GSexpression system in murine myeloma cells is described in Bebbington etal., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol.Prog. 11:1 (1995) which are incorporated in their entireties byreference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused to either the N-or C-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Antibodies of theinvention may also be fused to albumin (including but not limited torecombinant human serum albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is hereinincorporated by reference in its entirety. In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883 herein incorporated byreference in its entirety. Polynucleotides encoding fusion proteins ofthe invention are also encompassed by the invention. Such fusionproteins may, for example, facilitate purification and may increasehalf-life in vivo. Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2 or of the polypeptideencoded by the deposited clone may be fused or conjugated to the aboveantibody portions to increase the in vivo half life of the polypeptidesor for use in immunoassays using methods known in the art. Further, thepolypeptides corresponding to SEQ ID NO: 2 or to the polypeptide encodedby the deposited clone may be fused or conjugated to the above antibodyportions to facilitate purification. One reported example describeschimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc,^(99m)Tc), thallium (201Ti), gallium (68 Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ⁸⁸Re, ¹⁴²Pr, ₁₀₅Rh,and ⁹⁷Ru;

In specific embodiments, G-protein Chemokine Receptor (CCR5)polypeptides of the invention are attached to macrocyclic chelatorsuseful for conjugating radiometal ions, including but not limited to,¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferredembodiment, the radiometal ion associated with the macrocyclic chelatorsattached to G-protein Chemokine Receptor (CCR5) polypeptides of theinvention is ¹¹¹In. In another preferred embodiment, the radiometal ionassociated with the macrocyclic chelator attached to G-protein ChemokineReceptor (CCR5) polypeptides of the invention is ⁹⁰Y. In specificembodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the G-proteinChemokine Receptor (CCR5) polypeptide of the invention via a linkermolecule. Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art—see, for example, DeNardo etal., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug.Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.26(8):943-50, 1999 which are hereby incorporated by reference in theirentirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, whichdisclose chelating agents that may be conjugated to antibodies, andmethods for making and using them, are hereby incorporated by referencein their entireties. Though U.S. Pat. Nos. 5,652,361 and 5,756,065 focuson conjugating chelating agents to antibodies, one skilled in the artcould readily adapt the methods disclosed therein in order to conjugatechelating agents to other polypeptides.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The antibody conjugates of the invention can be used for modifying agiven biological response, the therapeutic agent or drug moiety is notto be construed as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies Forimmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as BIAcore analysis (see, e.g.,Example 59), FACS (Fluorescence activated cell sorter) analysis (see,e.g., Example 54), immunofluorescence (see, e.g., Example 56),immunocytochemistry, western blots (see Examples 64 and 65),radioimmunoassays, ELISA (enzyme linked immunosorbent assay) (See, e.g.,Example 54), “sandwich” immunoassays, immunoprecipitation assays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, to name but a few. Such assays are routine and well knownin the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety). Exemplaryimmunoassays are described briefly below (but are not intended by way oflimitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I), orfragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of unlabeled antigen, and the detectionof the antibody bound to the labeled antigen. The affinity of theantibody of interest for a G-protein Chemokine Receptor (CCR5) and thebinding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the G-protein Chemokine Receptor(CCR5) is incubated with antibody of interest conjugated to a labeledcompound (e.g., compound labeled with ³H or ¹²⁵I) in the presence ofincreasing amounts of an unlabeled second antibody. This kind ofcompetitive assay between two antibodies, may also be used to determineif two antibodies bind the same or different epitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies (including antibody fragmentsor variants thereof) to a G-protein Chemokine Receptor (CCR5), orfragments of a G-protein Chemokine Receptor (CCR5). BIAcore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized G-protein Chemokine Receptors (CCR5) ontheir surface as described in Example 59.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, andanti-retroviral agents (see Example 28, below). In a highly preferredembodiment, antibodies of the invention may be administered alone or incombination with and anti-retroviral agents (see Example 28, below).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸M or 10⁻⁸ M.Even more preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any G-protein Chemokine Receptor (CCR5) polypeptide can be used togenerate fusion proteins. For example, the G-protein Chemokine Receptor(CCR5) polypeptide, when fused to a second protein, can be used as anantigenic tag. Antibodies raised against the G-protein ChemokineReceptor (CCR5) polypeptide can be used to indirectly detect the secondprotein by binding to the G-protein Chemokine Receptor. Moreover,because secreted proteins target cellular locations based on traffickingsignals, the G-protein Chemokine Receptor (CCR5) polypeptides can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to G-protein Chemokine Receptor(CCR5) polypeptides include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

In certain preferred embodiments, G-protein Chemokine Receptor (CCR5)proteins of the invention comprise fusion proteins wherein the G-proteinChemokine Receptor (CCR5) polypeptides are those described above as m-n.In preferred embodiments, the application is directed to nucleic acidmolecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences encoding polypeptides having the amino acidsequence of the specific N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the G-protein Chemokine Receptor (CCR5) polypeptide.For instance, a region of additional amino acids, particularly chargedamino acids, may be added to the N-terminus of the G-protein ChemokineReceptor (CCR5) polypeptide to improve stability and persistence duringpurification from the host cell or subsequent handling and storage.Also, peptide moieties may be added to the G-protein Chemokine Receptor(CCR5) polypeptide to facilitate purification. Such regions may beremoved prior to final preparation of the G-protein Chemokine Receptor(CCR5) polypeptide. The addition of peptide moieties to facilitatehandling of polypeptides are familiar and routine techniques in the art.

As one of skill in the art will appreciate, polypeptides of the presentinvention and the epitope-bearing fragments thereof described above, canbe combined with heterologous polypeptide sequences. For example,polypeptides of the present invention (including fragments or variantsthereof), may be fused with the constant domain of immunoglobulins (IgA,IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combinationthereof and portions thereof, resulting in chimeric polypeptides. By wayof another non-limiting example, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) may be fusedwith albumin (including but not limited to recombinant human serumalbumin or fragments or variants thereof (see, e.g., U.S. Pat. No.5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No.5,766,883, issued Jun. 16, 1998, herein incorporated by reference intheir entirety)). In a preferred embodiment, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) are fused with the mature form of human serum albumin (i.e.,amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EPPatent 0 322 094) which is herein incorporated by reference in itsentirety. In another preferred embodiment, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) are fused with polypeptide fragments comprising, oralternatively consisting of, amino acid residues 1-z of human serumalbumin, where z is an integer from 369 to 419, as described in U.S.Pat. No. 5,766,883 herein incorporated by reference in its entirety.Polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) may be fused to either the N- orC-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Polynucleotidesencoding fusion proteins of the invention are also encompassed by theinvention.

These fusion proteins facilitate purification and show an increasedhalf-life in vivo. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature331:84-86 (1988).) Fusion proteins having disulfide-linked dimericstructures (due to the IgG) can also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the G-protein Chemokine Receptor (CCR5) polypeptides can befused to marker sequences, such as a peptide which facilitatespurification of G-protein Chemokine Receptor. In preferred embodiments,the marker amino acid sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,Chatsworth, Calif., 91311), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Another peptide tag useful forpurification, the “HA” tag, corresponds to an epitope derived from theinfluenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using the G-proteinChemokine Receptor (CCR5) polynucleotides or the polypeptides.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the G-proteinChemokine Receptor (CCR5) polynucleotide, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

G-protein Chemokine Receptor (CCR5) polynucleotides may be joined to avector containing a selectable marker for propagation in a host.Generally, a plasmid vector is introduced in a precipitate, such as acalcium phosphate precipitate, or in a complex with a charged lipid. Ifthe vector is a virus, it may be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

The G-protein Chemokine Receptor (CCR5) polynucleotide insert should beoperatively linked to an appropriate promoter, such as the phage lambdaPL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40early and late promoters and promoters of retroviral LTRs, to name afew. Other suitable promoters will be known to the skilled artisan. Theexpression constructs will further contain sites for transcriptioninitiation, termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the transcriptsexpressed by the constructs will preferably include a translationinitiating codon at the beginning and a termination codon (UAA, UGA orUAG) appropriately positioned at the end of the polypeptide to betranslated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418, glutamine synthase or neomycin resistance for eukaryotic cellculture and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris(ATCC Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, NSO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. The availability of drugswhich inhibit the function of the enzymes encoded by these selectablemarkers allows for selection of cell lines in which the vector sequenceshave been amplified after integration into the host cell's DNA. Anadvantage of glutamine synthase based vectors are the availability ofcell lines (e.g., the murine myeloma cell line, NSO) which are glutaminesynthase negative. Glutamine synthase expression systems can alsofunction in glutamine synthase expressing cells (e.g. Chinese HamsterOvary (CHO) cells) by providing additional inhibitor to prevent thefunctioning of the endogenous gene. Vectors that use glutamine synthaseas the selectable marker include the pEE6 expression vector described inStephens and Cockett, Nucl. Acids. Res 17:7110 (1989). A glutaminesynthase expression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorsthat may be used according to the present invention are commerciallyavailable from suppliers including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.). Expression and production of monoclonal antibodiesusing a GS expression system in murine myeloma cells is described inBebbington et al., Bio/technology 10:169 (1992) and in Biblia andRobinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated byreference.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that G-protein ChemokineReceptor (CCR5) polypeptides may in fact be expressed by a host celllacking a recombinant vector.

G-protein Chemokine Receptor (CCR5) polypeptides can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography (“HPLC”) is employedfor purification.

G-protein Chemokine Receptor (CCR5) polypeptides, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the G-proteinChemokine Receptor (CCR5) polypeptides may be glycosylated or may benon-glycosylated. In addition, G-protein Chemokine Receptor (CCR5)polypeptides may also include an initial modified methionine residue, insome cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to expressG-protein Chemokine Receptor (CCR5) protein in a eukaryotic system.Pichia pastoris is a methylotrophic yeast which can metabolize methanolas its sole carbon source. A main step in the methanol metabolizationpathway is the oxidation of methanol to formaldehyde using O₂. Thisreaction is catalyzed by the enzyme alcohol oxidase. In order tometabolize methanol as its sole carbon source, Pichia pastoris mustgenerate high levels of alcohol oxidase due, in part, to the relativelylow affinity of alcohol oxidase for O₂. Consequently, in a growth mediumdepending on methanol as a main carbon source, the promoter region ofone of the two alcohol oxidase genes (AOX1) is highly active. In thepresence of methanol, alcohol oxidase produced from the AOX1 genecomprises up to approximately 30% of the total soluble protein in Pichiapastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985);Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al.,Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous codingsequence, such as, for example, a G-protein Chemokine Receptor (CCR5)polynucleotide of the present invention, under the transcriptionalregulation of all or part of the AOX1 regulatory sequence is expressedat exceptionally high levels in Pichia yeast grown in the presence ofmethanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a G-protein Chemokine Receptor (CCR5) polypeptide of theinvention, as set forth herein, in a Pichea yeast system essentially asdescribed in “Pichia Protocols: Methods in Molecular Biology,” D. R.Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. Thisexpression vector allows expression and secretion of a G-proteinChemokine Receptor (CCR5) protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a G-protein Chemokine Receptor (CCR5)polynucleotide of the present invention, may be achieved by cloning theheterologous polynucleotide of the invention into an expression vectorsuch as, for example, pGAPZ or pGAPZalpha, and growing the yeast culturein the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., G-protein Chemokine Receptor (CCR5) codingsequence), and/or to include genetic material (e.g., heterologouspolynucleotide sequences) that is operably associated with G-proteinChemokine Receptor (CCR5) polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous G-protein ChemokineReceptor (CCR5) polynucleotides. For example, techniques known in theart may be used to operably associate heterologous control regions(e.g., promoter and/or enhancer) and endogenous G-protein ChemokineReceptor (CCR5) polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a G-protein ChemokineReceptor (CCR5) polypeptide can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the G-protein Chemokine Receptor (CCR5) polypeptidesequence. Non-classical amino acids include, but are not limited to, tothe D-isomers of the common amino acids, 2,4-diaminobutyric acid,a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,b-alanine, fluoro-amino acids, designer amino acids such as b-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

The invention encompasses G-protein Chemokine Receptor (CCR5)polypeptides which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The G-protein Chemokine Receptor (CCR5) polypeptides of the inventionmay be in monomers or multimers (i.e., dimers, trimers, tetramers andhigher multimers). Accordingly, the present invention relates tomonomers and multimers of the G-protein Chemokine Receptor (CCR5)polypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:2 orencoded by the HDGNR10 DNA contained in the deposited clone (includingfragments, variants, splice variants, and fusion proteins, correspondingto these as described herein). These homomers may contain G-proteinChemokine Receptor (CCR5) polypeptides having identical or differentamino acid sequences. In a specific embodiment, a homomer of theinvention is a multimer containing only G-protein Chemokine Receptor(CCR5) polypeptides having an identical amino acid sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingG-protein Chemokine Receptor (CCR5) polypeptides having different aminoacid sequences. In specific embodiments, the multimer of the inventionis a homodimer (e.g., containing G-protein Chemokine Receptor (CCR5)polypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing G-protein Chemokine Receptor (CCR5)polypeptides having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the G-protein Chemokine Receptor (CCR5)polypeptides of the invention. In a specific embodiment, the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the heteromeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the G-protein Chemokine Receptor (CCR5)polypeptides of the invention. Such covalent associations may involveone or more amino acid residues contained in the polypeptide sequence(e.g., that recited in SEQ ID NO:2, or contained in the polypeptideencoded by the clone HDGNR10). In one instance, the covalentassociations are cross-linking between cysteine residues located withinthe polypeptide sequences which interact in the native (i.e., naturallyoccurring) polypeptide. In another instance, the covalent associationsare the consequence of chemical or recombinant manipulation.Alternatively, such covalent associations may involve one or more aminoacid residues contained in the heterologous polypeptide sequence in aG-protein Chemokine Receptor (CCR5) fusion protein. In one example,covalent associations are between the heterologous sequence contained ina fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925).In a specific example, the covalent associations are between theheterologous sequence contained in a G-protein Chemokine Receptor-Fcfusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, oseteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Uses of the G-Protein Chemokine Receptor (CCR5) Polynucleotides

The G-protein Chemokine Receptor (CCR5) polynucleotides identifiedherein can be used in numerous ways as reagents. The followingdescription should be considered exemplary and utilizes knowntechniques.

There exists an ongoing need to identify new chromosome markers, sincefew chromosome marking reagents, based on actual sequence data (repeatpolymorphisms), are presently available.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:1 or fromthe deposited clone. Primers can be selected using computer analysis sothat primers do not span more than one predicted exon in the genomicDNA. These primers are then used for PCR screening of somatic cellhybrids containing individual human chromosomes. Only those hybridscontaining the human G-protein Chemokine Receptor (CCR5) genecorresponding to the SEQ ID NO:1 or to the deposited clone will yield anamplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the G-protein Chemokine Receptor (CCR5)polynucleotides can be achieved with panels of specific chromosomefragments. Other gene mapping strategies that can be used include insitu hybridization, prescreening with labeled flow-sorted chromosomes,and preselection by hybridization to construct chromosome specific-cDNAlibraries.

Precise chromosomal location of the G-protein Chemokine Receptor (CCR5)polynucleotides can also be achieved using fluorescence in situhybridization (FISH) of a metaphase chromosomal spread. This techniqueuses polynucleotides as short as 500 or 600 bases; however,polynucleotides 2,000-4,000 bp are preferred. For a review of thistechnique, see Verma et al., “Human Chromosomes: a Manual of BasicTechniques,” Pergamon Press, New York (1988).

For chromosome mapping, the G-protein Chemokine Receptor (CCR5)polynucleotides can be used individually (to mark a single chromosome ora single site on that chromosome) or in panels (for marking multiplesites and/or multiple chromosomes). Preferred polynucleotides correspondto the noncoding regions of the cDNAs or genomic clone because thecoding sequences are more likely conserved within gene families, thusincreasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in the G-proteinChemokine Receptor (CCR5) polynucleotide and the corresponding genebetween affected and unaffected individuals can be examined. First,visible structural alterations in the chromosomes, such as deletions ortranslocations, are examined in chromosome spreads or by PCR. If nostructural alterations exist, the presence of point mutations areascertained. Mutations observed in some or all affected individuals, butnot in normal individuals, indicates that the mutation may cause thedisease. However, complete sequencing of the G-protein ChemokineReceptor (CCR5) polypeptide and the corresponding gene from severalnormal individuals is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingG-protein Chemokine Receptor (CCR5) polynucleotides. Any of thesealterations (altered expression, chromosomal rearrangement, or mutation)can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anindividual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

In still another embodiment, the invention includes a kit for analyzingsamples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the present invention and a suitable container. In aspecific embodiment, the kit includes two polynucleotide probes definingan internal region of the polynucleotide of the present invention, whereeach probe has one strand containing a 31'mer-end internal to theregion. In a further embodiment, the probes may be useful as primers forpolymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or depressedpolynucleotide of the present invention expression will experience aworse clinical outcome relative to patients expressing the gene at alevel nearer the standard level.

By “measuring the expression level of polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of individuals nothaving a disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as semen, lymph,sera, plasma, urine, synovial fluid and spinal fluid) which contain thepolypeptide of the present invention, and other tissue sources found toexpress the polypeptide of the present invention. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

The method(s) provided above may preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingcancerous diseases and conditions. Such a method is described in U.S.Pat. Nos. 5,858,659 and 5,856,104. The U.S. Patents referenced supra arehereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the strong binding. In addition, it is morelikely that single base mismatches can be determined with PNA/DNAhybridization because a single mismatch in a PNA/DNA 15-mer lowers themelting point (T_(m)) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-merduplex. Also, the absence of charge groups in PNA means thathybridization can be done at low ionic strengths and reduce possibleinterference by salt during the analysis.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of pathological cellproliferative neoplasias which include, but are not limited to: acutemyelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Pathological cell proliferative disorders are often associated withinappropriate activation of proto-oncogenes. (Gelmann, E. P. et al.,“The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,”in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds.,161-182 (1985)). Neoplasias are now believed to result from thequalitative alteration of a normal cellular gene product, or from thequantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocyticleukemia cell line HL-60. When HL-60 cells are chemically induced tostop proliferation, the level of c-myc is found to be downregulated.(International Publication Number WO 91/15580) However, it has beenshown that exposure of HL-60 cells to a DNA construct that iscomplementary to the 5′ end of c-myc or c-myb blocks translation of thecorresponding mRNAs which downregulates expression of the c-myc or c-mybproteins and causes arrest of cell proliferation and differentiation ofthe treated cells. (International Publication Number WO 91/15580;Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al.,Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisanwould appreciate the present invention's usefulness would not be limitedto treatment of proliferative diseases, disorders, and/or conditions ofhematopoietic cells and tissues, in light of the numerous cells and celltypes of varying origins which are known to exhibit proliferativephenotypes.

In addition to the foregoing, a G-protein Chemokine Receptor (CCR5)polynucleotide can be used to control gene expression through triplehelix formation or antisense DNA or RNA. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression”, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). Both methods rely on binding of the polynucleotide to acomplementary DNA or RNA. For these techniques, preferredpolynucleotides are usually oligonucleotides 20 to 40 bases in lengthand complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

G-protein Chemokine Receptor (CCR5) polynucleotides are also useful ingene therapy. One goal of gene therapy is to insert a normal gene intoan organism having a defective gene, in an effort to correct the geneticdefect. G-protein Chemokine Receptor (CCR5) offers a means of targetingsuch genetic defects in a highly accurate manner. Another goal is toinsert a new gene that was not present in the host genome, therebyproducing a new trait in the host cell.

The G-protein Chemokine Receptor (CCR5) polynucleotides are also usefulfor identifying individuals from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique, an individual's genomic DNA is digested with one ormore restriction enzymes, and probed on a Southern blot to yield uniquebands for identifying personnel. This method does not suffer from thecurrent limitations of “Dog Tags” which can be lost, switched, orstolen, making positive identification difficult. The G-proteinChemokine Receptor (CCR5) polynucleotides can be used as additional DNAmarkers for RFLP.

The G-protein Chemokine Receptor (CCR5) polynucleotides can also be usedas an alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an individual's genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,individuals can be identified because each individual will have a uniqueset of DNA sequences. Once a unique ID database is established for anindividual, positive identification of that individual, living or dead,can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk,lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can beamplified using PCR. In one prior art technique, gene sequencesamplified from polymorphic loci, such as DQa class II HLA gene, are usedin forensic biology to identify individuals. (Erlich, H., PCRTechnology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,G-protein Chemokine Receptor (CCR5) polynucleotides can be used aspolymorphic markers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from G-protein Chemokine Receptor (CCR5) sequences.Panels of such reagents can identify tissue by species and/or by organtype. In a similar fashion, these reagents can be used to screen tissuecultures for contamination.

Because G-protein Chemokine Receptor (CCR5) is expressed in macrophagesand memory T cells, G-protein Chemokine Receptor (CCR5) polynucleotidesare useful as hybridization probes for differential identification ofthe tissue(s) or cell type(s) present in a biological sample. Similarly,polypeptides and antibodies directed to G-protein Chemokine Receptor(CCR5) polypeptides are useful to provide immunological probes fordifferential identification of the tissue(s) or cell type(s). Inaddition, for a number of diseases, disorders, and/or conditions of theabove tissues or cells, or in which these cells play a role,significantly higher or lower levels of G-protein Chemokine Receptor(CCR5) gene expression may be detected in certain tissues (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” G-protein Chemokine Receptor(CCR5) gene expression level, i.e., the G-protein Chemokine Receptor(CCR5) expression level in healthy tissue from an individual not havingthe immune system-related disorder.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves: (a) assaying G-protein Chemokine Receptor (CCR5) geneexpression level in cells or body fluid of an individual; (b) comparingthe G-protein Chemokine Receptor (CCR5) gene expression level with astandard G-protein Chemokine Receptor (CCR5) gene expression level,whereby an increase or decrease in the assayed G-protein ChemokineReceptor (CCR5) gene expression level compared to the standardexpression level is indicative of disorder in the immune system orrelated to the immune system.

In a further embodiment, the invention provides a method of usingG-protein Chemokine Receptor (CCR5) polynucleotides, or fragments orvariants thereof as a vaccine to elicit an immune response to G-proteinChemokine Receptor. In a preferred embodiment, the invention provides amethod of using G-protein Chemokine Receptor (CCR5) polynucleotides, orfragments or variants thereof as a DNA vaccine to elicit a humoral(antibody-mediated) immune response to G-protein Chemokine Receptor. Inother highly preferred embodiments, the invention provides a method ofusing G-protein Chemokine Receptor (CCR5) polynucleotides comprising thenucleotide sequence of one or more extracellular loops of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 89-102, 167-195 and/or261-274 of SEQ ID NO:2 or of the polypeptide encoded by the depositedclone (SEQ ID NO:22)) as a DNA vaccine to elicit immune response toG-protein Chemokine Receptor. In other highly preferred embodiments, theinvention provides a method of using G-protein Chemokine Receptor (CCR5)polynucleotides comprising the nucleotide sequence of the firstextracellular loop of G-protein Chemokine Receptor (CCR5) (i.e., aminoacids 89-102 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone (SEQ ID NO:22)) as a DNA vaccine to elicit an immuneresponse to G-protein Chemokine Receptor (CCR5). In other highlypreferred embodiments, the invention provides a method of usingG-protein Chemokine Receptor (CCR5) polynucleotides comprising thenucleotide sequence of the second extracellular loop of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 167-195 of SEQ ID NO:2 orof the polypeptide encoded by the deposited clone (SEQ ID NO:22)) as aDNA vaccine to elicit an immune response to G-protein Chemokine Receptor(CCR5). In other highly preferred embodiments, the invention provides amethod of using G-protein Chemokine Receptor (CCR5) polynucleotidescomprising the nucleotide sequence of the third extracellular loop ofG-protein Chemokine Receptor (CCR5) (i.e., 261-274 of SEQ ID NO:2 or ofthe polypeptide encoded by the deposited clone (SEQ ID NO:22)) as a DNAvaccine to elicit an immune response to G-protein Chemokine Receptor(CCR5).

In other highly preferred embodiments, the invention provides a methodof using G-protein Chemokine Receptor (CCR5) polynucleotides comprisingthe nucleotide sequence of one or more extracellular loops of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 89-102, 167-195 and/or261-274 of SEQ ID NO:2 or of the polypeptide encoded by the depositedclone (SEQ ID NO:22)) as a DNA vaccine to elicit a humoral immuneresponse to G-protein Chemokine Receptor. In other highly preferredembodiments, the invention provides a method of using G-proteinChemokine Receptor (CCR5) polynucleotides comprising the nucleotidesequence of the first extracellular loop of G-protein Chemokine Receptor(CCR5) (i.e., amino acids 89-102 of SEQ ID NO:2 or of the polypeptideencoded by the deposited clone (SEQ ID NO:22)) as a DNA vaccine toelicit a humoral immune response to G-protein Chemokine Receptor (CCR5).In other highly preferred embodiments, the invention provides a methodof using G-protein Chemokine Receptor (CCR5) polynucleotides comprisingthe nucleotide sequence of the second extracellular loop of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 167-195 of SEQ ID NO:2 orof the polypeptide encoded by the deposited clone (SEQ ID NO:22)) as aDNA vaccine to elicit a humoral immune response to G-protein ChemokineReceptor (CCR5). In other highly preferred embodiments, the inventionprovides a method of using G-protein Chemokine Receptor (CCR5)polynucleotides comprising the nucleotide sequence of the thirdextracellular loop of G-protein Chemokine Receptor (CCR5) (i.e., 261-274of SEQ ID NO:2 or of the polypeptide encoded by the deposited clone (SEQID NO:22)) as a DNA vaccine to elicit a humoral immune response toG-protein Chemokine Receptor (CCR5).

In another embodiment, the present invention provides a DNA vaccinecomprising, or alternatively consisting of, a G-protein ChemokineReceptor (CCR5) polynucleotide, or fragment or variant thereof. Inanother preferred embodiment, the present invention provides a DNAvaccine comprising, or alternatively consisting of, a G-proteinChemokine Receptor (CCR5) polynucleotide encoding the nucleotidesequence of one or more extracellular loops of G-protein ChemokineReceptor (CCR5) (i.e., amino acids 89-102, 167-195 and/or 261-274 of SEQID NO:2 or of the polypeptide encoded by the deposited clone (SEQ IDNO:22)). In another preferred embodiment, the present invention providesa DNA vaccine comprising, or alternatively consisting of, a G-proteinChemokine Receptor (CCR5) polynucleotide encoding the nucleotidesequence of the first extracellular loop of G-protein Chemokine Receptor(CCR5) (i.e., amino acids 89-102, of SEQ ID NO:2 or of the polypeptideencoded by the deposited clone (SEQ ID NO:22)). In another preferredembodiment, the present invention provides a DNA vaccine comprising, oralternatively consisting of, a G-protein Chemokine Receptor (CCR5)polynucleotide encoding the nucleotide sequence of the secondextracellular loop of G-protein Chemokine Receptor (CCR5) (i.e., aminoacids 167-195 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone (SEQ ID NO:22)). In another preferred embodiment, thepresent invention provides a DNA vaccine comprising, or alternativelyconsisting of, a G-protein Chemokine Receptor (CCR5) polynucleotideencoding the nucleotide sequence of the third extracellular loop ofG-protein Chemokine Receptor (CCR5) (i.e., amino acids 261-274 of SEQ IDNO:2 or of the polypeptide encoded by the deposited clone (SEQ IDNO:22)).

In highly preferred embodiments, the vaccines described above areadministered to an animal, including humans, to prevent viral infection.In even more highly preferred embodiments, the vaccines described aboveare administered to an animal, including humans, to prevent HIVinfection. In still other highly preferred embodiments, the vaccinesdescribed above are administered to an animal, including humans, toprevent poxvirus infection. In still other highly preferred embodiments,the vaccines described above are administered to an animal, includinghumans, to prevent cytomegalovirus infection.

In the very least, the G-protein Chemokine Receptor (CCR5)polynucleotides can be used as molecular weight markers on Southerngels, as diagnostic probes for the presence of a specific mRNA in aparticular cell type, as a probe to “subtract-out” known sequences inthe process of discovering novel polynucleotides, for selecting andmaking oligomers for attachment to a “gene chip” or other support, toraise anti-DNA antibodies using DNA immunization techniques, and as anantigen to elicit an immune response.

Uses of G-Protein Chemokine Receptor (CCRS) Polypeptides

G-protein Chemokine Receptor (CCR5) polypeptides can be used in numerousways. The following description should be considered exemplary andutilizes known techniques.

G-protein Chemokine Receptor (CCR5) polypeptides can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99 mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying G-protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99 mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of G-protein Chemokine Receptor(CCR5) polypeptide in cells or body fluid of an individual; (b)comparing the level of gene expression with a standard gene expressionlevel, whereby an increase or decrease in the assayed G-proteinChemokine Receptor (CCR5) polypeptide gene expression level compared tothe standard expression level is indicative of a disorder. With respectto cancer, the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

Moreover, G-protein Chemokine Receptor (CCR5) polypeptides can be usedto treat, prevent, and/or diagnose disease. For example, patients can beadministered G-protein Chemokine Receptor (CCR5) polypeptides in aneffort to replace absent or decreased levels of the G-protein ChemokineReceptor (CCR5) polypeptide (e.g., insulin), to supplement absent ordecreased levels of a different polypeptide (e.g., hemoglobin S forhemoglobin B, SOD, catalase, DNA repair proteins), to inhibit theactivity of a polypeptide (e.g., an oncogene or tumor suppressor), toactivate the activity of a polypeptide (e.g., by binding to a receptor),to reduce the activity of a membrane bound receptor by competing with itfor free ligand (e.g., soluble TNF receptors used in reducinginflammation), or to bring about a desired response (e.g., blood vesselgrowth inhibition, enhancement of the immune response to proliferativecells or tissues).

Similarly, antibodies directed to G-protein Chemokine Receptor (CCR5)polypeptides can also be used to treat, prevent, and/or diagnosedisease. For example, administration of an antibody directed to aG-protein Chemokine Receptor (CCR5) polypeptide can bind and reduceoverproduction of the polypeptide. Similarly, administration of anantibody can activate the polypeptide, such as by binding to apolypeptide bound to a membrane (receptor).

In a further embodiment, the invention provides a method of usingG-protein Chemokine Receptor (CCR5) polypeptides, or fragments orvariants as a vaccine to elicit an immune response to G-proteinChemokine Receptor. In a preferred embodiment, the invention provides amethod of using G-protein Chemokine Receptor (CCR5) polypeptides, orfragments or variants thereof as a vaccine to elicit a humoral(antibody-mediated) immune response to G-protein Chemokine Receptor. Inother highly preferred embodiments, the invention provides a method ofusing G-protein Chemokine Receptor (CCR5) polypeptides comprising theamino acid sequence of one or more extracellular loops of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 89-102, 167-195 and/or261-274 of SEQ ID NO:2 or of the polypeptide encoded by the depositedclone (SEQ ID NO:22)) as a vaccine to elicit an immune response toG-protein Chemokine Receptor. In other highly preferred embodiments, theinvention provides a method of using G-protein Chemokine Receptor (CCR5)polypeptides comprising the amino acid sequence of the firstextracellular loop of G-protein Chemokine Receptor (CCR5) (i.e., aminoacids 89-102 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone (SEQ ID NO:22)) as a vaccine to elicit an immuneresponse to G-protein Chemokine Receptor (CCR5). In other highlypreferred embodiments, the invention provides a method of usingG-protein Chemokine Receptor (CCR5) polypeptides comprising the aminoacid sequence of the second extracellular loop of G-protein ChemokineReceptor (CCR5) (i.e., amino acids 167-195 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone (SEQ ID NO:22)) as a vaccineto elicit an immune response to G-protein Chemokine Receptor (CCR5). Inother highly preferred embodiments, the invention provides a method ofusing G-protein Chemokine Receptor (CCR5) polypeptides comprising theamino acid sequence of the third extracellular loop of G-proteinChemokine Receptor (CCR5) (i.e., 261-274 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone (SEQ ID NO:22)) as a vaccineto elicit an immune response to G-protein Chemokine Receptor (CCR5).

In other highly preferred embodiments, the invention provides a methodof using G-protein Chemokine Receptor (CCR5) polypeptides comprising theamino acid sequence of one or more extracellular loops of G-proteinChemokine Receptor (CCR5) (i.e., amino acids 89-102, 167-195 and/or261-274 of SEQ ID NO:2 or of the polypeptide encoded by the depositedclone (SEQ ID NO:22)) as a vaccine to elicit a humoral immune responseto G-protein Chemokine Receptor. In other highly preferred embodiments,the invention provides a method of using G-protein Chemokine Receptor(CCR5) polypeptides comprising the amino acid sequence of the firstextracellular loop of G-protein Chemokine Receptor (CCR5) (i.e., aminoacids 89-102 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone (SEQ ID NO:22)) as a vaccine to elicit a humoral immuneresponse to G-protein Chemokine Receptor (CCR5). In other highlypreferred embodiments, the invention provides a method of usingG-protein Chemokine Receptor (CCR5) polypeptides comprising the aminoacid sequence of the second extracellular loop of G-protein ChemokineReceptor (CCR5) (i.e., amino acids 167-195 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone (SEQ ID NO:22)) as a vaccineto elicit a humoral immune response to G-protein Chemokine Receptor(CCR5). In other highly preferred embodiments, the invention provides amethod of using G-protein Chemokine Receptor (CCR5) polypeptidescomprising the amino acid sequence of the third extracellular loop ofG-protein Chemokine Receptor (CCR5) (i.e., 261-274 of SEQ ID NO:2 or ofthe polypeptide encoded by the deposited clone (SEQ ID NO:22)) as avaccine to elicit a humoral immune response to G-protein ChemokineReceptor (CCR5).

In another embodiment, the present invention provides a vaccinecomprising, or alternatively consisting of, a G-protein ChemokineReceptor (CCR5) polypeptide, or fragment or variant thereof. In anotherpreferred embodiment, the present invention provides a vaccinecomprising, or alternatively consisting of, a G-protein ChemokineReceptor (CCR5) polypeptide encoding the amino acid sequence of one ormore extracellular loops of G-protein Chemokine Receptor (CCR5) (i.e.,amino acids 89-102, 167-195 and/or 261-274 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone (SEQ ID NO:22)). In anotherpreferred embodiment, the present invention provides a vaccinecomprising, or alternatively consisting of, a G-protein ChemokineReceptor (CCR5) polypeptide encoding the amino acid sequence of thefirst extracellular loop of G-protein Chemokine Receptor (CCR5) (i.e.,amino acids 89-102, of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone (SEQ ID NO:22)). In another preferred embodiment, thepresent invention provides a vaccine comprising, or alternativelyconsisting of, a G-protein Chemokine Receptor (CCR5) polypeptideencoding the amino acid sequence of the second extracellular loop ofG-protein Chemokine Receptor (CCR5) (i.e., amino acids 167-195 of SEQ IDNO:2 or of the polypeptide encoded by the deposited clone (SEQ IDNO:22)). In another preferred embodiment, the present invention providesa vaccine comprising, or alternatively consisting of, a G-proteinChemokine Receptor (CCR5) polypeptide encoding the amino acid sequenceof the third extracellular loop of G-protein Chemokine Receptor (CCR5)(i.e., amino acids 261-274 of SEQ ID NO:2 or of the polypeptide encodedby the deposited clone (SEQ ID NO:22)).

In highly preferred embodiments, the vaccines described above areadministered to an animal, including humans, to prevent viral infection.In even more highly preferred embodiments, the vaccines described aboveare administered to an animal, including humans, to prevent HIVinfection. In still other highly preferred embodiments, the vaccinesdescribed above are administered to an animal, including humans, toprevent poxvirus infection. In still other highly preferred embodiments,the vaccines described above are administered to an animal, includinghumans, to prevent cytomegalovirus infection.

At the very least, the G-protein Chemokine Receptor (CCR5) polypeptidescan be used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. G-protein Chemokine Receptor (CCR5) polypeptides can also beused to raise antibodies, which in turn are used to measure proteinexpression from a recombinant cell, as a way of assessing transformationof the host cell. Moreover, G-protein Chemokine Receptor (CCR5)polypeptides can be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe G-protein Chemokine Receptor (CCR5) polypeptide of the presentinvention. This method requires a polynucleotide which codes for aG-protein Chemokine Receptor (CCR5) polypeptide operatively linked to apromoter and any other genetic elements necessary for the expression ofthe polypeptide by the target tissue. Such gene therapy and deliverytechniques are known in the art, see, for example, WO90/11092, which isherein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aG-protein Chemokine Receptor (CCR5) polynucleotide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, seeBelldegrun, A., et al., J. Natl Cancer Inst. 85: 207-216 (1993);Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini,M. et al., J. Immunology 153: 460-44615 (1994); Kaido, T., et al., Int.J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50:5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10(1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); andZhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which areherein incorporated by reference. In one embodiment, the cells which areengineered are arterial cells. The arterial cells may be reintroducedinto the patient through direct injection to the artery, the tissuessurrounding the artery, or through catheter injection.

As discussed in more detail below, the G-protein Chemokine Receptor(CCR5) polynucleotide constructs can be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, and the like). The G-protein Chemokine Receptor (CCR5)polynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the G-protein Chemokine Receptor (CCR5)polynucleotide is delivered as a naked polynucleotide. The term “naked”polynucleotide, DNA or RNA refers to sequences that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin or precipitating agents and the like. However,the G-protein Chemokine Receptor (CCR5) polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The G-protein Chemokine Receptorpolynucleotide vector constructs used inthe gene therapy method are preferably constructs that will notintegrate into the host genome nor will they contain sequences thatallow for replication. Appropriate vectors include pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG andpSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2available from Invitrogen. Other suitable vectors will be readilyapparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of G-protein Chemokine Receptor (CCR5)polynucleotide sequence. Suitable promoters include adenoviralpromoters, such as the adenoviral major late promoter; or heterologouspromoters, such as the cytomegalovirus (CMV) promoter; the respiratorysyncytial virus (RSV) promoter; inducible promoters, such as the MMTpromoter, the metallothionein promoter; heat shock promoters; thealbumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forG-protein Chemokine Receptor.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The G-protein Chemokine Receptor (CCR5) polynucleotide construct can bedelivered to the interstitial space of tissues within the an animal,including of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue. Interstitial space of thetissues comprises the intercellular, fluid, mucopolysaccharide matrixamong the reticular fibers of organ tissues, elastic fibers in the wallsof vessels or chambers, collagen fibers of fibrous tissues, or that samematrix within connective tissue ensheathing muscle cells or in thelacunae of bone. It is similarly the space occupied by the plasma of thecirculation and the lymph fluid of the lymphatic channels. Delivery tothe interstitial space of muscle tissue is preferred for the reasonsdiscussed below. They may be conveniently delivered by injection intothe tissues comprising these cells. They are preferably delivered to andexpressed in persistent, non-dividing cells which are differentiated,although delivery and expression may be achieved in non-differentiatedor less completely differentiated cells, such as, for example, stemcells of blood or skin fibroblasts. In vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked G-proteinChemokine Receptor (CCR5) DNA constructs can be delivered to arteriesduring angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the G-protein Chemokine Receptor (CCR5)polynucleotide constructs are complexed in a liposome preparation.Liposomal preparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner et al.,Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA (1989) 86:6077-6081, which is herein incorporated by reference); andpurified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192, which is herein incorporated by reference), infunctional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Feigneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingG-protein Chemokine Receptor. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding G-protein Chemokine Receptor. Suchretroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express G-protein Chemokine Receptor.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with G-protein Chemokine Receptor (CCR5) polynucleotide contained in anadenovirus vector. Adenovirus can be manipulated such that it encodesand expresses G-protein Chemokine Receptor (CCR5), and at the same timeis inactivated in terms of its ability to replicate in a normal lyticviral life cycle. Adenovirus expression is achieved without integrationof the viral DNA into the host cell chromosome, thereby alleviatingconcerns about insertional mutagenesis. Furthermore, adenoviruses havebeen used as live enteric vaccines for many years with an excellentsafety profile (Schwartz, A. R. et al. (1974) Am. Rev. Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer has beendemonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The G-protein ChemokineReceptor (CCR5) polynucleotide construct is inserted into the AAV vectorusing standard cloning methods, such as those found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989).The recombinant AAV vector is then transfected into packaging cellswhich are infected with a helper virus, using any standard technique,including lipofection, electroporation, calcium phosphate precipitation,etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses,vaccinia viruses, or herpes viruses. Once the packaging cells aretransfected and infected, they will produce infectious AAV viralparticles which contain the G-protein Chemokine Receptor (CCR5)polynucleotide construct. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the G-protein Chemokine Receptor (CCR5)polynucleotide construct integrated into its genome, and will expressG-protein Chemokine Receptor.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding G-protein Chemokine Receptor) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the G-protein Chemokine Receptor (CCR5) desiredendogenous polynucleotide sequence so the promoter will be operablylinked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous G-protein ChemokineReceptor (CCR5) sequence is placed under the control of the promoter.The promoter then drives the expression of the endogenous G-proteinChemokine Receptor (CCR5) sequence.

The polynucleotides encoding G-protein Chemokine Receptor (CCR5) may beadministered along with other polynucleotides encoding an angiogenicprotein. Examples of angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3,epidermal growth factor alpha and beta, platelet-derived endothelialcell growth factor, platelet-derived growth factor, tumor necrosisfactor alpha, hepatocyte growth factor, insulin like growth factor,colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

In one preferred embodiment, the polynucleotide encoding G-proteinChemokine Receptor (CCR5) contains a secretory signal sequence thatfacilitates secretion of the protein. Typically, the signal sequence ispositioned in the coding region of the polynucleotide to be expressedtowards or at the 5′ end of the coding region. The signal sequence maybe homologous or heterologous to the polynucleotide of interest and maybe homologous or heterologous to the cells to be transfected.Additionally, the signal sequence may be chemically synthesized usingmethods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities of G-protein Chemokine Receptor

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), can beused in assays to test for one or more biological activities. IfG-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), doexhibit activity in a particular assay, it is likely that G-proteinChemokine Receptor (CCR5) may be involved in the diseases associatedwith the biological activity. Therefore, G-protein Chemokine Receptor(CCR5) could be used to treat, prevent, and/or diagnose the associateddisease.

Ligands of the G-protein Chemokine Receptor (CCR5) include MIP-1alpha,MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, RANTES, and Eotaxin. TheG-protein Chemokine Receptor (CCR5) is also a major co-receptor for HIV,and may be also be recognized by other infectious agents, such as otherviruses, to allow entry into the cell. Thus, G-protein ChemokineReceptor (CCR5) polynucleotides, polypeptides, agonists and antagoniststhereof are useful for treating, preventing and diagnosing diseasesassociated with any of the above ligands, such as the diseases disclosedherein. In highly preferred embodiments, G-protein Chemokine Receptor(CCR5) polynucleotides, polypeptides, agonists and antagonists thereofare useful for treating, preventing and diagnosing HIV infection and/orconditions associated with HIV infection, as described in the sectionentitled “Treatment and prevention of HIV Infection.”

G-protein Chemokine Receptor (CCR5) is predominantly expressed onmonocytes and T-cells. Expression of G-protein Chemokine Receptor (CCR5)is found on microglial, dendritic and some hematopoietic stem cells.Activation of G-protein Chemokine Receptor (CCR5) on macrophages andlymphocytes by G-protein Chemokine Receptor (CCR5) ligands (especially,RANTES, MIP-1beta and MIP-1alpha) primarily results in chemoattractionof these cell types to sites of inflammation, often sites of infection.G-protein Chemokine Receptor (CCR5) may also be involved in theinduction of chemotaxis in NK cells, eosinophils and basophils.Activation of G-protein Chemokine Receptor (CCR5) on macrophages andlymphocytes by G-protein Chemokine Receptor (CCR5) ligands (especially,RANTES, MIP-1beta and MIP-1alpha) can promote interactions betweenT-cells and antigen presenting cells (e.g., dendritic cells, macrophagesand B cells.) G-protein Chemokine Receptor (CCR5) may also be involvedin cell sticking and migration through blood vessels via adhesionmolecules in transit to site of inflammation. Accordingly, compositionsof the invention (including polynucleotides, polypeptides and antibodiesof the invention, and fragments and variants thereof) may be used in thediagnosis, prognosis, prevention, and/or treatment of diseases and/ordisorders associated with defects in the biological activities ofG-protein Chemokine Receptor (CCR5) such as those described above.

In preferred embodiments, compositions of the invention (includingpolynucleotides, polypeptides and antibodies of the invention, andfragments and variants thereof) may be used in the diagnosis, prognosis,prevention, and/or treatment of diseases and/or disorders relating toimmune function (e.g., viral infection (especially HIV infection,poxvirus infection and/or cytomegalovirus infection); autoimmunediseases (such as Rheumatoid Arthritis, Grave's disease and MultipleSclerosis); immune cell chemotaxis; inflammatory conditions; and/or asdescribed in “Immune Activity”) and neoplastic disorders such as thosedescribed under “Hyperproliferative Disorders” below).

G-protein Chemokine Receptor (CCR5) polynucleotides, polypeptides,agonists and antagonists (including antibodies) of the invention areuseful in the diagnosis, prognosis, prevention, and/or treatment ofdiseases and/or disorders associated with activities that include, butare not limited to, immune cell chemoattraction, immune cell activation,antigen presentation, inflammation, and viral infection.

More generally, G-protein Chemokine Receptor (CCR5) polynucleotides,polypeptides, agonists and antagonists (including antibodies) of theinvention may be useful for the diagnosis, prognosis, prevention, and/ortreatment of diseases and/or disorders described below.

Treatment and Prevention of HIV Infection. As CCR5 is HIV co-receptorfor macrophage tropic HIV it has major impact on HIV infection anddisease progression, especially early in HIV infection when HIV ispredominantly of R5 macrophage-tropic strains. Therefore, G-proteinChemokine Receptor (CCR5) polynucleotides or polypeptides, or agonists(including antibodies) or antagonists (including antibodies) ofG-protein Chemokine Receptor (CCR5), may be used to diagnose, treat,prevent, and/or ameliorate HIV infection.

In specific embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or amelioratediseases, disorders or conditions associated with HIV infection.Conditions associated with HI infection include, but are not limited to,Pneumocystis carinii pneumonia, Wasting syndrome, Kaposi's sarcoma,Esophageal candidiasis, and pulmonary Candidiasis, disseminated orextrapulmonary Mycobacterium avium-intracellulare complex, disseminatedor extrapulmonary Mycobacterium kansasii, Cytomegalovirus disease,Cytomegalovirus retinitis, HIV encephalopathy, Herpes simplex disease,extrapulmonary Cryptococcosis, Toxoplasmosis of brain, chronicCryptosporidiosis, chronic intestinal Cryptosporidiosis, immunoblasticlymphoma, extrapulmonary Mycobacterium tuberculosis, pulmonaryMycobacterium tuberculosis, Mycobacterial disease, extrapulmonaryMycobacteridl disease, Burkitt's lymphoma, progressive multifocalleukoencephalopathy, primary brain lymphoma, chronic Isosporiasis,chronic intestinal Isosporiasis, disseminated or extrapulmonaryCoccidioidomycosis, Salmonella septicemia, multiple or recurrentbacterial infections, invasive cervical carcinoma, disseminated orextrapulmonary Histoplasmosis, Lymphoid interstitial pneumonia,pulmonary lymphoid hyperplasia, recurrent pneumonia, severeimmunosuppression and/or AIDS dementia.

In preferred embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorateopportunistic infections (e.g., Herpes virus infection, MycobacteriuniTuberculosis infection, or cytomegalovirus infection) associated withHIV infection.

In preferred embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorateopportunistic Pneumocystis carinji infection associated with HIVinfection.

In preferred embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorateKaposi's sarcoma associated with HIV infection.

In other preferred embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorate theearly stages of HIV infection.

In other embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorate thelate stages of HIV infection.

In other embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5), may be used to diagnose, treat, prevent, and/or ameliorate thelate stages of HIV infection.

In still other embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5) are used as a prophylatic to prevent HIV infection in persons whohave an HIV-infected sexual partner or persons with reason to believethey have been exposed to HIV, (e.g., persons who have been stuck with aneedle that had previously been in contact with the biological fluid ofanother individual (or animal), or rape victims).

In still other embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists (including antibodies) orantagonists (including antibodies) of G-protein Chemokine Receptor(CCR5) are used as a prophylatic to prevent maternal-fetal transmissionof HIV.

Immune Activity. G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), may be useful in treating diseases, disorders, and/or conditionsof the immune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through a process called hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune diseases, disorders, and/or conditions may be genetic,somatic, such as cancer or some autoimmune diseases, disorders, and/orconditions, acquired (e.g., by chemotherapy or toxins), or infectious.Moreover, G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), can be used as a marker or detector of a particular immunesystem disease or disorder.

The G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the present invention and/or agonists or antagoniststhereof may be used to modulate hematopoietic activity (the formation ofblood cells). For example, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be used to increase the quantity ofall or subsets of blood cells, such as, for example, erythrocytes,lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils,neutrophils, mast cells, macrophages) and platelets. The ability todecrease the quantity of blood cells or subsets of blood cells may beuseful in the prevention, detection, diagnosis and/or treatment ofanemias and leukopenias described below. Alternatively, the G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of thepresent invention and/or agonists or antagonists thereof may be used todecrease the quantity of all or subsets of blood cells, such as, forexample, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of leukocytoses, such as, for exampleeosinophilia.

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), may beuseful in treating, preventing, and/or diagnosing diseases, disorders,and/or conditions of hematopoietic cells. G-protein Chemokine Receptor(CCR5) polynucleotides or polypeptides, or agonists or antagonists ofG-protein Chemokine Receptor (CCR5), could be used to increasedifferentiation and proliferation of hematopoietic cells, including thepluripotent stem cells, in an effort to treat or prevent those diseases,disorders, and/or conditions associated with a decrease in certain (ormany) types hematopoietic cells. Examples of immunologic deficiencysyndromes include, but are not limited to, blood protein diseases,disorders, and/or conditions (e.g. agammaglobulinemia,dysgammaglobulinemia), ataxia telangiectasia, common variableimmunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,leukocyte adhesion deficiency syndrome, lymphopenia, phagocytebactericidal dysfunction, severe combined immunodeficiency (SCIDs),Wiskott-Aldrich Disorder, anemia, thrombocytopenia, leukopenia,neutropenia, anemia or hemoglobinuria. Alternatively, G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of theinvention, and/or agonists or antagonists thereof could be used toincrease differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat or preventthose diseases, disorders, and/or conditions associated with an increasein certain (or many) types of hematopoietic cells, including but notlimited to, histiocytosis.

In another embodiment, a G-protein Chemokine Receptor (CCR5) polypeptideof the invention, or polynucleotides, antibodies, agonists, orantagonists corresponding to that G-protein Chemokine Receptor (CCR5)polypeptide, may be used to treat diseases and disorders of the immunesystem and/or to inhibit or enhance an immune response generated bycells associated with the tissue(s) in which the polypeptide of theinvention is expressed.

G-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the invention, and/or agonists or antagonists thereof may be usefulin treating, preventing, diagnosing, and/or prognosingimmunodeficiencies, including both congenital and acquiredimmunodeficiencies. Examples of B cell immunodeficiencies in whichimmunoglobulin levels B cell function and/or B cell numbers aredecreased include: X-linked agammaglobulinemia (Bruton's disease),X-linked infantile agammaglobulinemia, X-linked immunodeficiency withhyper IgM, non X-linked immunodeficiency with hyper IgM, X-linkedlymphoproliferative syndrome (XLP), agammaglobulinemia includingcongenital and acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessiveagammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgAdeficiency, selective IgG subclass deficiencies, IgG subclass deficiency(with or without IgA deficiency), Ig deficiency with increased IgM, IgGand IgA deficiency with increased IgM, antibody deficiency with normalor elevated Igs, Ig heavy chain deletions, kappa chain deficiency, Bcell lymphoproliferative disorder (BLPD), common variableimmunodeficiency (CVID), common variable immunodeficiency (CVI)(acquired), and transient hypogammaglobulinemia of infancy.

In specific embodiments, ataxia-telangiectasia or conditions associatedwith ataxia-telangiectasia are treated, prevented, diagnosed, and/orprognosing using the polypeptides or polynucleotides of the invention,and/or agonists or antagonists thereof.

Examples of congenital immunodeficiencies in which T cell and/or B cellfunction and/or number is decreased include, but are not limited to:DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including,but not limited to, X-linked SCID, autosomal recessive SCID, adenosinedeaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency,Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrichsyndrome, and ataxia telangiectasia), thymic hypoplasia, third andfourth pharyngeal pouch syndrome, 22q11.2 deletion, chronicmucocutaneous candidiasis, natural killer cell deficiency (NK),idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant Tcell defect (unspecified), and unspecified immunodeficiency of cellmediated immunity.

In specific embodiments, DiGeorge anomaly or conditions associated withDiGeorge anomaly are treated, prevented, diagnosed, and/or prognosedusing polypeptides or polynucleotides of the invention, or antagonistsor agonists thereof.

Other immunodeficiencies that may be treated, prevented, diagnosed,and/or prognosed using polypeptides or polynucleotides of the invention,and/or agonists or antagonists thereof, include, but are not limited to,chronic granulomatous disease, Chédiak-Higashi syndrome, myeloperoxidasedeficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency,X-linked lymphoproliferative syndrome (XLP), leukocyte adhesiondeficiency, complement component deficiencies (including C1, C2, C3, C4,C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymicalymphoplasia-aplasia, immunodeficiency with thymoma, severe congenitalleukopenia, dysplasia with immunodeficiency, neonatal neutropenia, shortlimbed dwarfism, and Nezelof syndrome-combined immunodeficiency withIgs.

In a preferred embodiment, the immunodeficiencies and/or conditionsassociated with the immunodeficiencies recited above are treated,prevented, diagnosed and/or prognosed using G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides of the invention, and/oragonists or antagonists thereof.

In a preferred embodiment G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof could be used as an agent to boostimmunoresponsiveness among immunodeficient individuals. In specificembodiments, G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the invention, and/or agonists or antagonists thereofcould be used as an agent to boost immunoresponsiveness among B celland/or T cell immunodeficient individuals.

Moreover, G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), can also be used to modulate hemostatic (the stopping ofbleeding) or thrombolytic activity (clot formation). For example, byincreasing hemostatic or thrombolytic activity, G-protein ChemokineReceptor (CCR5) polynucleotides or polypeptides, or agonists orantagonists of G-protein Chemokine Receptor (CCR5), could be used totreat or prevent blood coagulation diseases, disorders, and/orconditions (e.g., afibrinogenemia, factor deficiencies), blood plateletdiseases, disorders, and/or conditions (e.g. thrombocytopenia), orwounds resulting from trauma, surgery, or other causes. Alternatively,G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), that candecrease hemostatic or thrombolytic activity could be used to inhibit ordissolve clotting. These molecules could be important in the treatmentor prevention of heart attacks (infarction), strokes, or scarring.

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), may alsobe useful in treating, preventing, and/or diagnosing autoimmunediseases, disorders, and/or conditions. Many autoimmune diseases,disorders, and/or conditions result from inappropriate recognition ofself as foreign material by immune cells. This inappropriate recognitionresults in an immune response leading to the destruction of the hosttissue. Therefore, the administration of G-protein Chemokine Receptor(CCR5) polynucleotides or polypeptides, or agonists or antagonists ofG-protein Chemokine Receptor (CCR5), that can inhibit an immuneresponse, particularly the proliferation, differentiation, or chemotaxisof T-cells, may be an effective therapy in preventing autoimmunediseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by G-proteinChemokine Receptor (CCR5) include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by G-protein Chemokine Receptor(CCR5) polynucleotides or polypeptides, or agonists or antagonists ofG-protein Chemokine Receptor. Moreover, these molecules can be used totreat anaphylaxis, hypersensitivity to an antigenic molecule, or bloodgroup incompatibility.

Additionally, G-protein Chemokine Receptor (CCR5) polypeptides orpolynucleotides of the invention, and/or agonists or antagoniststhereof, may be used to treat, prevent, diagnose and/or prognoseIgE-mediated allergic reactions. Such allergic reactions include, butare not limited to, asthma, rhinitis, and eczema. In specificembodiments, G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the invention, and/or agonists or antagonists thereofmay be used to modulate IgE concentrations in vitro or in vivo.

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), may alsobe used to treat, prevent, and/or diagnose organ rejection orgraft-versus-host disease (GVHD). Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists or antagonists of G-proteinChemokine Receptor (CCR5), that inhibits an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in preventing organ rejection orGVHD. In specific embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing experimental allergic and hyperacutexenograft rejection.

Similarly, G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), may also be used to modulate inflammation. For example, sincepolypeptides, antibodies, or polynucleotides of the invention, and/oragonists or antagonists of the invention may inhibit the activation,proliferation and/or differentiation of cells involved in aninflammatory response, these molecules can be used to prevent and/ortreat chronic and acute inflammatory conditions. Such inflammatoryconditions include, but are not limited to, for example, inflammationassociated with infection (e.g., septic shock, sepsis, or systemicinflammatory response syndrome), ischemia-reperfusion injury, endotoxinlethality, complement-mediated hyperacute rejection, nephritis, cytokineor chemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, over production of cytokines (e.g., TNF or IL-1), respiratorydisorders (e.g., asthma and allergy); gastrointestinal disorders (e.g.,inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung,bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis;ischemic brain injury and/or stroke, traumatic brain injury,neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer'sdisease); AIDS-related dementia; and prion disease); cardiovasculardisorders (e.g., atherosclerosis, myocarditis, cardiovascular disease,and cardiopulmonary bypass complications); as well as many additionaldiseases, conditions, and disorders that are characterized byinflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma,pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusioninjury, Grave's disease, systemic lupus erythematosus, diabetesmellitus, and allogenic transplant rejection).

Because inflammation is a fundamental defense mechanism, inflammatorydisorders can effect virtually any tissue of the body. Accordingly,polynucleotides, polypeptides, and antibodies of the invention, as wellas agonists or antagonists thereof, have uses in the treatment oftissue-specific inflammatory disorders, including, but not limited to,adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis,blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis,cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis,dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis,eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis,gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis,laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis,mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis,orchitis, osteochondritis, otitis, pericarditis, peritendonitis,peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis,pulpitis, retinitis, rhinitis, salpingitis, scleritis,sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis,stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis,and vaginitis.

In other embodiments, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be useful as an agent to enhance themigration, phagocytosis, superoxide production, antibody dependentcellular cytotoxicity of neutrophils, eosionophils and macrophages.

In another embodiment, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be useful in diagnosing, prognosing,preventing, and/or treating diseases and disorders characterized by orassociated with increased or decreased numbers of white blood cells.Leukopenia occurs when the number of white blood cells decreases belownormal. Leukopenias include, but are not limited to, neutropenia andlymphocytopenia. An increase in the number of white blood cells comparedto normal is known as leukocytosis. The body generates increased numbersof white blood cells during infection. Thus, leukocytosis may simply bea normal physiological parameter that reflects infection. Alternatively,leukocytosis may be an indicator of injury or other disease such ascancer. Leokocytoses, include but are not limited to, eosinophilia, andaccumulations of macrophages. In specific embodiments, the G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of thepresent invention and/or agonists or antagonists thereof may be usefulin diagnosing, prognosing, preventing, and/or treating leukopenia. Inother specific embodiments, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be useful in diagnosing, prognosing,preventing, and/or treating leukocytosis.

Leukopenia may be a generalized decreased in all types of white bloodcells, or may be a specific depletion of particular types of white bloodcells. Thus, in specific embodiments, the G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides of the present inventionand/or agonists or antagonists thereof may be useful in diagnosing,prognosing, preventing, and/or treating decreases in neutrophil numbers,known as neutropenia. Neutropenias that may be diagnosed, prognosed,prevented, and/or treated by the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof include, but are not limited to,infantile genetic agranulocytosis, familial neutropenia, cyclicneutropenia, neutropenias resulting from or associated with dietarydeficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency),neutropenias resulting from or associated with drug treatments (e.g.,antibiotic regimens such as penicillin treatment, sulfonamide treatment,anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, andcancer chemotherapy), and neutropenias resulting from increasedneutrophil destruction that may occur in association with some bacterialor viral infections, allergic disorders, autoimmune diseases, conditionsin which an individual has an enlarged spleen (e.g., Felty syndrome,malaria and sarcoidosis), and some drug treatment regimens.

The G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the present invention and/or agonists or antagoniststhereof may be useful in diagnosing, prognosing, preventing, and/ortreating lymphocytopenias (decreased numbers of B and/or T lymphocytes),including, but not limited lymphocytopenias resulting from or associatedwith stress, drug treatments (e.g., drug treatment with corticosteroids,cancer chemotherapies, and/or radiation therapies), AIDS and/or otherdiseases such as, for example, cancer, rheumatoid arthritis, systemiclupus erythematosus, chronic infections, some viral infections and/orhereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndrome,severe combined immunodeficiency, ataxia telangiectasia).

The G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the present invention and/or agonists or antagoniststhereof may be useful in diagnosing, prognosing, preventing, and/ortreating diseases and disorders associated with macrophage numbersand/or macrophage function including, but not limited to, Gaucher'sdisease, Niemann-Pick disease, Letterer-Siwe disease andHand-Schuller-Christian disease.

In another embodiment, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be useful in diagnosing, prognosing,preventing, and/or treating diseases and disorders associated witheosinophil numbers and/or eosinophil function including, but not limitedto, idiopathic hypereosinophilic syndrome, eosinophilia-myalgiasyndrome, and Hand-Schuller-Christian disease.

In yet another embodiment, the G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof may be useful in diagnosing, prognosing,preventing, and/or treating leukemias and lymphomas including, but notlimited to, acute lymphocytic (lymphoblastic) leukemia (ALL), acutemyeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic)leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cellleukemias, Sezary syndrome, and Hairy cell leukemia), chronic myelocytic(myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.

In other embodiments, polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, are useful todiagnose, prognose, prevent, and/or treat immune complex diseases,including, but not limited to, serum sickness, post streptococcalglomerulonephritis, polyarteritis nodosa, and immune complex-inducedvasculitis.

Polypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the invention can be used to treat, detect, and/or prevent infectiousagents. For example, by increasing the immune response, particularlyincreasing the proliferation activation and/or differentiation of Band/or T cells, infectious diseases may be treated, detected, and/orprevented. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, G-protein Chemokine Receptor (CCR5) polynucleotidesand/or polypeptides of the invention, and/or agonists or antagoniststhereof may also directly inhibit the infectious agent (refer to sectionof application listing infectious agents, etc), without necessarilyeliciting an immune response.

In another embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a vaccine adjuvant that enhances immuneresponsiveness to an antigen. In a specific embodiment, G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of theinvention, and/or agonists or antagonists thereof are used as anadjuvant to enhance tumor-specific immune responses.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an adjuvant to enhance anti-viral immuneresponses. Anti-viral immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include virus and virusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions of the invention areused as an adjuvant to enhance an immune response to a virus, disease,or symptom selected from the group consisting of: AIDS, meningitis,Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specificembodiment, the compositions of the invention are used as an adjuvant toenhance an immune response to a virus, disease, or symptom selected fromthe group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue,rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza,measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever,herpes simplex, and yellow fever.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an adjuvant to enhance anti-bacterial oranti-fungal immune responses. Anti-bacterial or anti-fungal immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include bacteria or fungus and bacteria or fungusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions of the invention areused as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:tetanus, Diphtheria, botulism, and meningitis type B.

In another specific embodiment, the compositions of the invention areused as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group Bstreptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, and Borrelia burgdorferi.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an adjuvant to enhance anti-parasiticimmune responses. Anti-parasitic immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include parasiteand parasite associated diseases or symptoms described herein orotherwise known in the art. In specific embodiments, the compositions ofthe invention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria) or Leishmania.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may also be employed to treat infectious diseasesincluding silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; forexample, by preventing the recruitment and activation of mononuclearphagocytes.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an antigen for the generation ofantibodies to inhibit or enhance immune mediated responses againstpolypeptides of the invention.

In one embodiment, G-protein Chemokine Receptor (CCR5) polynucleotidesand/or polypeptides of the invention, and/or agonists or antagoniststhereof are administered to an animal (e.g., mouse, rat, rabbit,hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow,sheep, dog, cat, non-human primate, and human, most preferably human) toboost the immune system to produce increased quantities of one or moreantibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinityantibody production and immunoglobulin class switching (e.g., IgG, IgA,IgM, and IgE), and/or to increase an immune response.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a stimulator of B cell responsiveness topathogens.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an activator of T cells.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent that elevates the immune statusof an individual prior to their receipt of immunosuppressive therapies.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to induce higher affinityantibodies.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to increase serumimmunoglobulin concentrations.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to accelerate recovery ofimmunocompromised individuals.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to boost immunoresponsivenessamong aged populations and/or neonates.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an immune system enhancer prior to,during, or after bone marrow transplant and/or other transplants (e.g.,allogeneic or xenogeneic organ transplantation). With respect totransplantation, compositions of the invention may be administered priorto, concomitant with, and/or after transplantation. In a specificembodiment, compositions of the invention are administered aftertransplantation, prior to the beginning of recovery of T-cellpopulations. In another-specific embodiment, compositions of theinvention are first administered after transplantation after thebeginning of recovery of T cell populations, but prior to full recoveryof B cell populations.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to boost immunoresponsivenessamong individuals having an acquired loss of B cell function. Conditionsresulting in an acquired loss of B cell function that may be amelioratedor treated by administering the polypeptides, antibodies,polynucleotides and/or agonists or antagonists thereof, include, but arenot limited to, HIV Infection, AIDS, bone marrow transplant, and B cellchronic lymphocytic leukemia (CLL).

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to boost immunoresponsivenessamong individuals having a temporary immune deficiency. Conditionsresulting in a temporary immune deficiency that may be ameliorated ortreated by administering the polypeptides, antibodies, polynucleotidesand/or agonists or antagonists thereof, include, but are not limited to,recovery from viral infections (e.g., influenza), conditions associatedwith malnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, and recovery from surgery.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a regulator of antigen presentation bymonocytes, dendritic cells, and/or B-cells. In one embodiment, G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of theinvention, and/or agonists or antagonists thereof enhance antigenpresentation or antagonizes antigen presentation in vitro or in vivo.Moreover, in related embodiments, said enhancement or antagonism ofantigen presentation may be useful as an anti-tumor treatment or tomodulate the immune system.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as an agent to direct an individual'simmune system towards development of a humoral response (i.e. TH2) asopposed to a TH1 cellular response.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a means to induce tumor proliferationand thus make it more susceptible to anti-neoplastic agents. Forexample, multiple myeloma is a slowly dividing disease and is thusrefractory to virtually all anti-neoplastic regimens. If these cellswere forced to proliferate more rapidly their susceptibility profilewould likely change.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a stimulator of B cell production inpathologies such as AIDS, chronic lymphocyte disorder and/or CommonVariable Immunodificiency.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a therapy for generation and/orregeneration of lymphoid tissues following surgery, trauma or geneticdefect. In another specific embodiment, G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides of the invention, and/oragonists or antagonists thereof are used in the pretreatment of bonemarrow samples prior to transplant.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a gene-based therapy for geneticallyinherited disorders resulting in immuno-incompetence/immunodeficiencysuch as observed among SCID patients.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a means of activatingmonocytes/macrophages to defend against parasitic diseases that effectmonocytes such as Leishmania.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a means of regulating secreted cytokinesthat are elicited by polypeptides of the invention.

In another embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used in one or more of the applicationsdescribed herein, as they may apply to veterinary medicine.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a means of blocking various aspects ofimmune responses to foreign agents or self. Examples of diseases orconditions in which blocking of certain aspects of immune responses maybe desired include autoimmune disorders such as lupus, and arthritis, aswell as immunoresponsiveness to skin allergies, inflammation, boweldisease, injury and diseases/disorders associated with pathogens.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a therapy for preventing the B cellproliferation and Ig secretion associated with autoimmune diseases suchas idiopathic thrombocytopenic purpura, systemic lupus erythematosus andmultiple sclerosis.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a inhibitor of B and/or T cell migrationin endothelial cells. This activity disrupts tissue architecture orcognate responses and is useful, for example in disrupting immuneresponses, and blocking sepsis.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a therapy for chronichypergammaglobulinemia evident in such diseases as monoclonal gammopathyof undetermined significance (MGUS), Waldenstrom's disease, relatedidiopathic monoclonal gammopathies, and plasmacytomas.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may be employed for instance to inhibit polypeptidechemotaxis and activation of macrophages and their precursors, and ofneutrophils, basophils, B lymphocytes and some T-cell subsets, e.g.,activated and CD8 cytotoxic T cells and natural killer cells, in certainautoimmune and chronic inflammatory and infective diseases. Examples ofautoimmune diseases are described herein and include multiple sclerosis,and insulin-dependent diabetes.

The G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the invention, and/or agonists or antagonists thereofmay also be employed to treat idiopathic hyper-eosinophilic syndrome by,for example, preventing eosinophil production and migration.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used to enhance or inhibit complement mediatedcell lysis.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used to enhance or inhibit antibody dependentcellular cytotoxicity.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may also be employed for treating atherosclerosis,for example, by preventing monocyte infiltration in the artery wall.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may be employed to treat adult respiratory distresssyndrome (ARDS).

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may be useful for stimulating wound and tissuerepair, stimulating angiogenesis, and/or stimulating the repair ofvascular or lymphatic diseases or disorders. Additionally, agonists andantagonists of the invention may be used to stimulate the regenerationof mucosal surfaces.

In a specific embodiment, polynucleotides or polypeptides, and/oragonists thereof are used to diagnose, prognose, treat, and/or prevent adisorder characterized by primary or acquired immunodeficiency,deficient serum immunoglobulin production, recurrent infections, and/orimmune system dysfunction. Moreover, polynucleotides or polypeptides,and/or agonists thereof may be used to treat or prevent infections ofthe joints, bones, skin, and/or parotid glands, blood-borne infections(e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),autoimmune diseases (e.g., those disclosed herein), inflammatorydisorders, and malignancies, and/or any disease or disorder or conditionassociated with these infections, diseases, disorders and/ormalignancies) including, but not limited to, CVID, other primary immunedeficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitismedia, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster(e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseasesand disorders that may be prevented, diagnosed, prognosed, and/ortreated with polynucleotides or polypeptides, and/or agonists of thepresent invention include, but are not limited to, HIV infection,HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunctionanemia, thrombocytopenia, and hemoglobinuria.

In another embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used to treat, and/or diagnose an individualhaving common variable immunodeficiency disease (“CVID”; also known as“acquired agammaglobulinemia” and “acquired hypogammaglobulinemina”) ora subset of this disease.

In a specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof may be used to diagnose, prognose, prevent, and/ortreat cancers or neoplasms including immune cell or immunetissue-related cancers or neoplasms. Examples of cancers or neoplasmsthat may be prevented, diagnosed, or treated by G-protein ChemokineReceptor (CCR5) polynucleotides and/or polypeptides of the invention,and/or agonists or antagonists thereof include, but are not limited to,acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin'sdisease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chroniclymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt'slymphoma, EBV-transformed diseases, and/or diseases and disordersdescribed in the section entitled “Hyperproliferative Disorders”elsewhere herein.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a therapy for decreasing cellularproliferation of Large B-cell Lymphomas.

In another specific embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are used as a means of decreasing the involvement ofB cells and Ig associated with Chronic Myelogenous Leukemia.

In specific embodiments, the compositions of the invention are used asan agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy.

Antagonists of the invention include, for example, binding and/orinhibitory antibodies, antisense nucleic acids, ribozymes or solubleforms of the polypeptides of the present invention (e.g., Fc fusionprotein). Agonists of the invention include, for example, binding orstimulatory antibodies, and soluble forms of the polypeptides (e.g., Fcfusion proteins). G-protein Chemokine Receptor (CCR5) polynucleotidesand/or polypeptides of the invention, and/or agonists or antagoniststhereof may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as described herein.

In another embodiment, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof are administered to an animal (including, but notlimited to, those listed above, and also including transgenic animals)incapable of producing functional endogenous antibody molecules orhaving an otherwise compromised endogenous immune system, but which iscapable of producing human immunoglobulin molecules by means of areconstituted or partially reconstituted immune system from anotheranimal (see, e.g., published PCT Application Nos. WO98/24893,WO/9634096, WO/9633735, and WO/9110741). Administration of G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of theinvention, and/or agonists or antagonists thereof to such animals isuseful for the generation of monoclonal antibodies against the G-proteinChemokine Receptor (CCR5) polynucleotides and/or polypeptides of theinvention, and/or agonists or antagonists thereof.

Chemotaxis. G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), may have chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells,mast cells, eosinophils, epithelial and/or endothelial cells) to aparticular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), mayincrease chemotaxic activity of particular cells. These chemotacticmolecules can then be used to treat, prevent, and/or diagnoseinflammation, infection, hyperproliferative diseases, disorders, and/orconditions, or any immune system disorder by increasing the number ofcells targeted to a particular location in the body. For example,chemotaxic molecules can be used to treat, prevent, and/or diagnosewounds and other trauma to tissues by attracting immune cells to theinjured location. Chemotactic molecules of the present invention canalso attract fibroblasts, which can be used to treat, prevent, and/ordiagnose wounds.

It is also contemplated that G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists or antagonists of G-proteinChemokine Receptor (CCR5), may inhibit chemotactic activity. Thesemolecules could also be used to treat, prevent, and/or diagnosediseases, disorders, and/or conditions. Thus, G-protein ChemokineReceptor (CCR5) polynucleotides or polypeptides, or agonists orantagonists of G-protein Chemokine Receptor (CCR5), could be used as aninhibitor of chemotaxis.

Infectious Disease. G-protein Chemokine Receptor (CCR5) polynucleotidesor polypeptides, or agonists or antagonists of G-protein ChemokineReceptor (CCR5), can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, G-protein Chemokine Receptor (CCR5) polynucleotides orpolypeptides, or agonists or antagonists of G-protein Chemokine Receptor(CCR5), may also directly inhibit the infectious agent, withoutnecessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited tothe following DNA and RNA viruses and viral families: Arbovirus,Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such asSmallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae(HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus).Viruses falling within these families can cause a variety of diseases orsymptoms, including, but not limited to: arthritis, bronchiollitis,respiratory syncytial virus, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunisticinfections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the commoncold, Polio, leukemia, Rubella, sexually transmitted diseases, skindiseases (e.g., Kaposi's, warts), and viremia. Polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedto treat, prevent, and/or diagnose any of these symptoms or diseases. Inspecific embodiments, polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat: meningitis, Dengue, EBV,and/or hepatitis (e.g., hepatitis B). In an additional specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat patients nonresponsive to one or moreother commercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

In highly preferred embodiments, G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides of the present invention and/oragonists or antagonists thereof, are used to diagnose, treat, prevent orameliorate HIV infection.

In other highly preferred embodiments, G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides of the present inventionand/or agonists or antagonists thereof, are used to diagnose, treat,prevent or ameliorate Cytomegalovirus infections.

In other highly preferred embodiments, G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides of the present inventionand/or agonists or antagonists thereof, are used to diagnose, treat,prevent or ameliorate Poxyiridae infections.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi. Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat: tetanus, Diptheria, botulism, and/or meningitis typeB.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. Polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Neurological Diseases. Nervous system diseases, disorders, and/orconditions, which can be treated with the G-protein Chemokine Receptor(CCR5) compositions of the invention (e.g., G-protein Chemokine Receptor(CCR5) polypeptides, polynucleotides, and/or agonists or antagonists),include, but are not limited to, nervous system injuries, and diseases,disorders, and/or conditions which result in either a disconnection ofaxons, a diminution or degeneration of neurons, or demyelination.Nervous system lesions which may be treated in a patient (includinghuman and non-human mammalian patients) according to the invention,include but are not limited to, the following lesions of either thecentral (including spinal cord, brain) or peripheral nervous systems:(1) ischemic lesions, in which a lack of oxygen in a portion of thenervous system results in neuronal injury or death, including cerebralinfarction or ischemia, or spinal cord infarction or ischemia; (2)traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries; (3) malignant lesions, inwhich a portion of the nervous system is destroyed or injured bymalignant tissue which is either a nervous system associated malignancyor a malignancy derived from non-nervous system tissue; (4) infectiouslesions, in which a portion of the nervous system is destroyed orinjured as a result of infection, for example, by an abscess orassociated with infection by human immunodeficiency virus, herpeszoster, or herpes simplex virus or with Lyme disease, tuberculosis,syphilis; (5) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis (ALS); (6) lesions associated with nutritionaldiseases, disorders, and/or conditions, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including but not limited to, vitamin B12deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the G-protein Chemokine Receptor (CCR5)polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the G-protein ChemokineReceptor (CCR5) compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the G-protein ChemokineReceptor (CCR5) polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseneural cell injury associated with cerebral ischemia. In another aspectof this embodiment, the G-protein Chemokine Receptor (CCR5)polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction.

In another aspect of this embodiment, the G-protein Chemokine Receptor(CCR5) polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with a stroke. In another aspect of this embodiment, theG-protein Chemokine Receptor (CCR5) polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose cerebral neural cell injury associated with a stroke.

In a further aspect of this embodiment, the G-protein Chemokine Receptor(CCR5) polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with a heart attack. In another aspect of this embodiment,the G-protein Chemokine Receptor (CCR5) polypeptides, polynucleotides,or agonists or antagonists of the invention are used to treat, prevent,and/or diagnose cerebral neural cell injury associated with a heartattack.

The compositions of the invention which are useful for treating,preventing, and/or diagnosing a nervous system disorder may be selectedby testing for biological activity in promoting the survival ordifferentiation of neurons. For example, and not by way of limitation,G-protein Chemokine Receptor (CCR5) compositions of the invention whichelicit any of the following effects may be useful according to theinvention: (1) increased survival time of neurons in culture; (2)increased sprouting of neurons in culture or in vivo; (3) increasedproduction of a neuron-associated molecule in culture or in vivo, e.g.,choline acetyltransferase or acetylcholinesterase with respect to motorneurons; or (4) decreased symptoms of neuron dysfunction in vivo. Sucheffects may be measured by any method known in the art. In preferred,non-limiting embodiments, increased survival of neurons may routinely bemeasured using a method set forth herein or otherwise known in the art,such as, for example, the method set forth in Arakawa et al. (J.Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may bedetected by methods known in the art, such as, for example, the methodsset forth in Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown etal. (Ann. Rev. Neurosci. 4:17-42 (1981)); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., using techniquesknown in the art and depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated according to the invention include, butare not limited to, diseases, disorders, and/or conditions such asinfarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as diseases,disorders, and/or conditions that selectively affect neurons such asamyotrophic lateral sclerosis, and including, but not limited to,progressive spinal muscular atrophy, progressive bulbar palsy, primarylateral sclerosis, infantile and juvenile muscular atrophy, progressivebulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis andthe post polio syndrome, and Hereditary Motorsensory Neuropathy(Charcot-Marie-Tooth Disease).

Further, G-protein Chemokine Receptor (CCR5) polypeptides orpolynucleotides of the invention may play a role in neuronal survival;synapse formation; conductance; neural differentiation, etc. Thus,compositions of the invention (including G-protein Chemokine Receptor(CCR5) polynucleotides and/or polypeptides, and/or agonists orantagonists thereof) may be used to diagnose and/or treat or preventdiseases or disorders associated with these roles, including, but notlimited to, learning and/or cognition disorders. The compositions of theinvention may also be useful in the treatment or prevention ofneurodegenerative disease states and/or behavioral disorders. Suchneurodegenerative disease states and/or behavioral disorders include,but are not limited to, Alzheimer's Disease, Parkinson's Disease,Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses, autism, and altered behaviors, includingdisorders in feeding, sleep patterns, balance, and perception. Inaddition, compositions of the invention may also play a role in thetreatment, prevention and/or detection of developmental disordersassociated with the developing embryo, or sexually-linked disorders.

Additionally, G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides, and/or agonists or antagonists thereof, may be useful inprotecting neural cells from diseases, damage, disorders, or injury,associated with cerebrovascular disorders including, but not limited to,carotid artery diseases (e.g., carotid artery thrombosis, carotidstenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebralaneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, orWallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subduralhematoma, or subarachnoid hemorrhage), cerebral infarction, cerebralischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome,or vertebrobasilar insufficiency), vascular dementia (e.g.,multi-infarct), leukomalacia, periventricular, and vascular headache(e.g., cluster headache or migraines).

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing G-protein Chemokine Receptor (CCR5)polynucleotides and/or polypeptides, and/or agonists or antagoniststhereof, for therapeutic purposes, for example, to stimulateneurological cell proliferation and/or differentiation. Therefore,polynucleotides, polypeptides, agonists and/or antagonists of theinvention may be used to treat and/or detect neurologic diseases.Moreover G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides, and/or agonists or antagonists thereof, can be used as amarker or detector of a particular nervous system disease or disorder.

Examples of neurologic diseases which can be treated or detected withthe G-protein Chemokine Receptor (CCR5) polynucleotides and/orpolypeptides of the present invention, and/or agonists or antagoniststhereof, include brain diseases, such as metabolic brain diseases whichincludes phenylketonuria such as maternal phenylketonuria, pyruvatecarboxylase deficiency, pyruvate dehydrogenase complex deficiency,Wernicke's Encephalopathy, brain edema, brain neoplasms such ascerebellar neoplasms which include infratentorial neoplasms, cerebralventricle neoplasms such as choroid plexus neoplasms, hypothalamicneoplasms, supratentorial neoplasms, canavan disease, cerebellardiseases such as cerebellar ataxia which include spinocerebellardegeneration such as ataxia telangiectasia, cerebellar dyssynergia,Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellaratrophy, cerebellar neoplasms such as infratentorial neoplasms, diffusecerebral sclerosis such as encephalitis periaxialis, globoid cellleukodystrophy, metachromatic leukodystrophy and subacute sclerosingpanencephalitis.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include cerebrovascular disorders (such as carotid artery diseases whichinclude carotid artery thrombosis, carotid stenosis and MoyamoyaDisease), cerebral amyloid angiopathy, cerebral aneurysm, cerebralanoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,cerebral artery diseases, cerebral embolism and thrombosis such ascarotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome,cerebral hemorrhage such as epidural hematoma, subdural hematoma andsubarachnoid hemorrhage, cerebral infarction, cerebral ischemia such astransient cerebral ischemia, Subclavian Steal Syndrome andvertebrobasilar insufficiency, vascular dementia such as multi-infarctdementia, periventricular leukomalacia, vascular headache such ascluster headache and migraine.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include dementia such as AIDS Dementia Complex, presenile dementia suchas Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementiasuch as Alzheimer's Disease and progressive supranuclear palsy, vasculardementia such as multi-infarct dementia, encephalitis which includeencephalitis periaxialis, viral encephalitis such as epidemicencephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borneencephalitis and West Nile Fever, acute disseminated encephalomyelitis,meningoencephalitis such as uveomeningoencephalitic syndrome,Postencephalitic Parkinson Disease and subacute sclerosingpanencephalitis, encephalomalacia such as periventricular leukomalacia,epilepsy such as generalized epilepsy which includes infantile spasms,absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome,tonic-clonic epilepsy, partial epilepsy such as complex partialepilepsy, frontal lobe epilepsy and temporal lobe epilepsy,post-traumatic epilepsy, status epilepticus such as Epilepsia PartialisContinua, and Hallervorden-Spatz Syndrome.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include hydrocephalus such as Dandy-Walker Syndrome and normal pressurehydrocephalus, hypothalamic diseases such as hypothalamic neoplasms,cerebral malaria, narcolepsy which includes cataplexy, bulbarpoliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome,thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma andZellweger Syndrome, central nervous system infections such as AIDSDementia Complex, Brain Abscess, subdural empyema, encephalomyelitissuch as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis,Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include meningitis such as arachnoiditis, aseptic meningtitis such asviral meningtitis which includes lymphocytic choriomeningitis, Bacterialmeningtitis which includes Haemophilus Meningtitis, ListeriaMeningtitis, Meningococcal Meningtitis such as Waterhouse-FriderichsenSyndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungalmeningitis such as Cryptococcal Meningtitis, subdural effusion,meningoencephalitis such as uvemeningoencephalitic syndrome, myelitissuch as transverse myelitis, neurosyphilis such as tabes dorsalis,poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitissyndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, BovineSpongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru,Scrapie), and cerebral toxoplasmosis.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include central nervous system neoplasms such as brain neoplasms thatinclude cerebellar neoplasms such as infratentorial neoplasms, cerebralventricle neoplasms such as choroid plexus neoplasms, hypothalamicneoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cordneoplasms which include epidural neoplasms, demyelinating diseases suchas Canavan Diseases, diffuse cerebral sceloris which includesadrenoleukodystrophy, encephalitis periaxialis, globoid cellleukodystrophy, diffuse cerebral sclerosis such as metachromaticleukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagicencephalomyelitis, progressive multifocal leukoencephalopathy, multiplesclerosis, central pontine myelinolysis, transverse myelitis,neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome,Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseasessuch as amyotonia congenita, amyotrophic lateral sclerosis, spinalmuscular atrophy such as Werdnig-Hoffmann Disease, spinal cordcompression, spinal cord neoplasms such as epidural neoplasms,syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardationsuch as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome,Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), SandhoffDisease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystica and spina bifida occulta.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include hereditary motor and sensory neuropathies which includeCharcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease,hereditary spastic paraplegia, Werdnig-Hoffmann Disease, HereditarySensory and Autonomic Neuropathies such as Congenital Analgesia andFamilial Dysautonomia, Neurologic manifestations (such as agnosia thatinclude Gerstmann's Syndrome, Amnesia such as retrograde amnesia,apraxia, neurogenic bladder, cataplexy, communicative disorders such ashearing disorders that includes deafness, partial hearing loss, loudnessrecruitment and tinnitus, language disorders such as aphasia whichinclude agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexiasuch as Acquired Dyslexia, language development disorders, speechdisorders such as aphasia which includes anomia, broca aphasia andWernicke Aphasia, articulation disorders, communicative disorders suchas speech disorders which include dysarthria, echolalia, mutism andstuttering, voice disorders such as aphonia and hoarseness, decerebratestate, delirium, fasciculation, hallucinations, meningism, movementdisorders such as angelman syndrome, ataxia, athetosis, chorea,dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis andtremor, muscle hypertonia such as muscle rigidity such as stiff-mansyndrome, muscle spasticity, paralysis such as facial paralysis whichincludes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, opthalmoplegiasuch as diplopia, Duane's Syndrome, Horner's Syndrome, Chronicprogressive external opthalmoplegia such as Kearns Syndrome, BulbarParalysis, Tropical Spastic Paraparesis, Paraplegia such asBrown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocalcord paralysis, paresis, phantom limb, taste disorders such as ageusiaand dysgeusia, vision disorders such as amblyopia, blindness, colorvision defects, diplopia, hemianopsia, scotoma and subnormal vision,sleep disorders such as hypersomnia which includes Kleine-LevinSyndrome, insomnia, and somnambulism, spasm such as trismus,unconsciousness such as coma, persistent vegetative state and syncopeand vertigo, neuromuscular diseases such as amyotonia congenita,amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motorneuron disease, muscular atrophy such as spinal muscular atrophy,Charcot-Marie Disease and Werdnig-Hoffmann Disease, PostpoliomyelitisSyndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica,Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis,Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-ManSyndrome, peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autononuc nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, opthalmoplegia such asDuane's Syndrome, Horner's Syndrome, Chronic Progressive ExternalOpthalmoplegia which includes Kearns Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica and Swayback, and Diabeticneuropathies such as diabetic foot.

Additional neurologic diseases which can be treated or detected with theG-protein Chemokine Receptor (CCR5) polynucleotides and/or polypeptidesof the present invention, and/or agonists or antagonists thereof,include nerve compression syndromes such as carpal tunnel syndrome,tarsal tunnel syndrome, thoracic outlet syndrome such as cervical ribsyndrome, ulnar nerve compression syndrome, neuralgia such as causalgia,cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia,neuritis such as experimental allergic neuritis, optic neuritis,polyneuritis, polyradiculoneuritis and radiculities such aspolyradiculitis, hereditary motor and sensory neuropathies such asCharcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease,Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, HereditarySensory and Autonomic Neuropathies which include Congenital Analgesiaand Familial Dysautonoria, POEMS Syndrome, Sciatica, Gustatory Sweatingand Tetany).

Hyperproliferative Disorders. G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists or antagonists of G-proteinChemokine Receptor (CCR5), can be used to treat, prevent, and/ordiagnose hyperproliferative diseases, disorders, and/or conditions,including neoplasms. G-protein Chemokine Receptor (CCR5) polynucleotidesor polypeptides, or agonists or antagonists of G-protein ChemokineReceptor (CCR5), may inhibit the proliferation of the disorder throughdirect or indirect interactions. Alternatively, G-protein ChemokineReceptor (CCR5) polynucleotides or polypeptides, or agonists orantagonists of G-protein Chemokine Receptor (CCR5), may proliferateother cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by G-protein ChemokineReceptor (CCR5) polynucleotides or polypeptides, or agonists orantagonists of G-protein Chemokine Receptor (CCR5), include, but are notlimited to neoplasms located in the: colon, abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by G-proteinChemokine Receptor (CCR5) polynucleotides or polypeptides, or agonistsor antagonists of G-protein Chemokine Receptor. Examples of suchhyperproliferative diseases, disorders, and/or conditions include, butare not limited to: hypergammaglobulinemia, lymphoproliferativediseases, disorders, and/or conditions, paraproteinemias, purpura,sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher'sDisease, histiocytosis, and any other hyperproliferative disease,besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cellproliferative diseases, disorders, and/or conditions by inserting intoan abnormally proliferating cell a polynucleotide of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative diseases, disorders, and/or conditions inindividuals comprising administration of one or more active gene copiesof the present invention to an abnormally proliferating cell or cells.In a preferred embodiment, polynucleotides of the present invention is aDNA construct comprising a recombinant expression vector effective inexpressing a DNA sequence encoding said polynucleotides. In anotherpreferred embodiment of the present invention, the DNA constructencoding the polynucleotides of the present invention is inserted intocells to be treated utilizing a retrovirus, or more preferably anadenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, whichis hereby incorporated by reference). In a most preferred embodiment,the viral vector is defective and will not transform non-proliferatingcells, only proliferating cells. Moreover, in a preferred embodiment,the polynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating one ormore of the described diseases, disorders, and/or conditions. Methodsfor producing anti-polypeptides and anti-polynucleotide antibodiespolyclonal and monoclonal antibodies are described in detail elsewhereherein. Such antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing cellproliferative and/or differentiation diseases, disorders, and/orconditions as described herein. Such treatment comprises administering asingle or multiple doses of the antibody, or a fragment, derivative, ora conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diseases, disorders, and/orconditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M.Even more preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Moreover, polypeptides of the present invention are useful in inhibitingthe angiogenesis of proliferative cells or tissues, either alone, as aprotein fusion, or in combination with other polypeptides directly orindirectly, as described elsewhere herein. In a most preferredembodiment, said anti-angiogenesis effect may be achieved indirectly,for example, through the inhibition of hematopoietic, tumor-specificcells, such as tumor-associated macrophages (See Joseph I B, et al. JNatl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated byreference). Antibodies directed to polypeptides or polynucleotides ofthe present invention may also result in inhibition of angiogenesisdirectly, or indirectly (See Witte L, et al., Cancer Metastasis Rev.17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including G-protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat Res 400(1-2):447-55(1998), Med Hypotheses. 50(5):423-33 (1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int J TissueReact; 20(1):3-15 (1998), which are all hereby incorporated byreference).

Polypeptides, including G-protein fusions to, or fragments thereof, ofthe present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such therapeuticaffects of the present invention may be achieved either alone, or incombination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders. G-protein Chemokine Receptor (CCR5)polynucleotides or polypeptides, or agonists or antagonists of G-proteinChemokine Receptor (CCR5), encoding G-protein Chemokine Receptor (CCR5)may be used to treat, prevent, and/or diagnose cardiovascular diseases,disorders, and/or conditions, including peripheral artery disease, suchas limb ischemia.

Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaun ay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

G-protein Chemokine Receptor (CCR5) polynucleotides or polypeptides, oragonists or antagonists of G-protein Chemokine Receptor (CCR5), areespecially effective for the treatment of critical limb ischemia andcoronary disease.

G-protein Chemokine Receptor (CCR5) polypeptides may be administeredusing any method known in the art, including, but not limited to, directneedle injection at the delivery site, intravenous injection, topicaladministration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. G-protein ChemokineReceptor (CCR5) polypeptides may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringG-protein Chemokine Receptor (CCR5) polynucleotides are described inmore detail herein.

Treatment of Carbohydrate Metabolism Disorders. In specific embodiments,the polynucleotides and/or polypeptides corresponding to this geneand/or agonists or antagonists of those polypeptides (includingantibodies) as well as fragments and variants of those polynucleotides,polypeptides, agonists and antagonists, may be used to diagnose,prognose, treat, prevent, or ameliorate diseases and disordersassociated with aberrant glucose metabolism or glucose uptake intocells.

In a specific embodiment, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists and/or antagonists thereofmay be used to diagnose, prognose, treat, prevent, and/or amelioratetype I diabetes mellitus (insulin dependent diabetes mellitus, IDDM).

In another embodiment, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists and/or antagonists thereofmay be used to diagnose, prognose, treat, prevent, and/or amelioratetype II diabetes mellitus (insulin resistant diabetes mellitus).

Additionally, in other embodiments, the polynucleotides and/orpolypeptides corresponding to this gene and/or antagonists thereof(especially neutralizing or antagonistic antibodies) may be used todiagnose, prognose, treat, prevent, and/or ameliorate conditionsassociated with (type I or type II) diabetes mellitus, including, butnot limited to, diabetic ketoacidosis, diabetic coma, nonketotichyperglycemic-hyperosmolar coma, seizures, mental confusion, drowsiness,cardiovascular disease (e.g., heart disease, atherosclerosis,microvascular disease, hypertension, stroke, and other diseases anddisorders as described in the “Cardiovascular Disorders” section),dyslipidemia, kidney disease (e.g., renal failure and nephropathy) nervedamage, neuropathy, vision impairment (e.g., diabetic retinopathy andblindness), ulcers and impaired wound healing, infections (e.g.,infectious diseases and disorders as described in the “InfectiousDiseases” section, especially of the urinary tract and skin), carpaltunnel syndrome and Dupuytren's contracture.

In other embodiments, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists or antagonists thereof areadministered to an animal, preferably a mammal, and most preferably ahuman, in order to regulate the animal's weight. In specific embodimentsthe polynucleotides and/or polypeptides corresponding to this geneand/or agonists or antagonists thereof are administered to an animal,preferably a mammal, and most preferably a human, in order to controlthe animal's weight by modulating a biochemical pathway involvinginsulin. In still other embodiments the polynucleotides and/orpolypeptides corresponding to this gene and/or agonists or antagoniststhereof are administered to an animal, preferably a mammal, and mostpreferably a human, in order to control the animal's weight bymodulating a biochemical pathway involving insulin-like growth factor.

Anti-Angiogenesis Activity. The naturally occurring balance betweenendogenous stimulators and inhibitors of angiogenesis is one in whichinhibitory influences predominate. Rastinejad et al., Cell 56:345-355(1989). In those rare instances in which neovascularization occurs undernormal physiological conditions, such as wound healing, organregeneration, embryonic development, and female reproductive processes,angiogenesis is stringently regulated and spatially and temporallydelimited. Under conditions of pathological angiogenesis such as thatcharacterizing solid tumor growth, these regulatory controls fail.Unregulated angiogenesis becomes pathologic and sustains progression ofmany neoplastic and non-neoplastic diseases. A number of seriousdiseases are dominated by abnormal neovascularization including solidtumor growth and metastases, arthritis, some types of eye diseases,disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moseset al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med.,333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411(1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse,Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol.94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In anumber of pathological conditions, the process of angiogenesiscontributes to the disease state. For example, significant data haveaccumulated which suggest that the growth of solid tumors is dependenton angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases, disorders,and/or conditions associated with neovascularization by administrationof the polynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)) Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat or prevent a cancer or tumor. Cancers which may betreated with polynucleotides, polypeptides, antagonists and/or agonistsinclude, but are not limited to solid tumors, including prostate, lung,breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, thyroid cancer; primary tumors and metastases;melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-smallcell lung cancer; colorectal cancer; advanced malignancies; and bloodborn tumors such as leukemias. For example, polynucleotides,polypeptides, antagonists and/or agonists may be delivered topically, inorder to treat or prevent cancers such as skin cancer, head and necktumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonistsand/or agonists may be utilized to treat, prevent, and/or diagnosesuperficial forms of bladder cancer by, for example, intravesicaladministration. Polynucleotides, polypeptides, antagonists and/oragonists may be delivered directly into the tumor, or near the tumorsite, via injection or a catheter. Of course, as the artisan of ordinaryskill will appreciate, the appropriate mode of administration will varyaccording to the cancer to be treated. Other modes of delivery arediscussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be usefulin treating other diseases, disorders, and/or conditions, besidescancers, which involve angiogenesis. These diseases, disorders, and/orconditions include, but are not limited to: benign tumors, for examplehemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenicgranulomas; artheroscleric plaques; ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormalblood vessel growth) of the eye; rheumatoid arthritis; psoriasis;delayed wound healing; endometriosis; vasculogenesis; granulations;hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma;vascular adhesions; myocardial angiogenesis; coronary collaterals;cerebral collaterals; arteriovenous malformations; ischemic limbangiogenesis; Osler-Webber Syndrome; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; fibromusculardysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating neovascular diseases of theeye, including for example, corneal neovascularization, neovascularglaucoma, proliferative diabetic retinopathy, retrolental fibroplasiaand macular degeneration.

Moreover, ocular diseases, disorders, and/or conditions associated withneovascularization which can be treated with the polynucleotides andpolypeptides of the present invention (including agonists and/orantagonists) include, but are not limited to: neovascular glaucoma,diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of diseases, disorders, and/orconditions can result in corneal neovascularization, including forexample, corneal infections (e.g., trachoma, herpes simplex keratitis,leishmaniasis and onchocerciasis), immunological processes (e.g., graftrejection and Stevens-Johnson's syndrome), alkali burns, trauma,inflammation (of any cause), toxic and nutritional deficiency states,and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. In one embodiment, the compoundmay be administered topically to the eye in order to treat or preventearly forms of neovascular glaucoma. Within other embodiments, thecompound may be implanted by injection into the region of the anteriorchamber angle. Within other embodiments, the compound may also be placedin any location such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, diseases, disorders, and/or conditions which can betreated with the polynucleotides, polypeptides, agonists and/or agonistsinclude, but are not limited to, hemangioma, arthritis, psoriasis,angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, whichcan be treated with be treated with the polynucleotides, polypeptides,agonists and/or agonists include, but are not limited to, solid tumors,blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma,benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis,psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the presentinvention may be incorporated into surgical sutures in order to preventstitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilizedin a wide variety of surgical procedures. For example, within one aspectof the present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Binding Activity. G-protein Chemokine Receptor (CCR5) polypeptides maybe used to screen for molecules that bind to G-protein ChemokineReceptor (CCR5) or for molecules to which G-protein Chemokine Receptor(CCR5) binds. The binding of G-protein Chemokine Receptor (CCR5) and themolecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the G-protein Chemokine Receptor (CCR5) or themolecule bound. Examples of such molecules include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand ofG-protein Chemokine Receptor (CCR5), e.g., a fragment of the ligand, ora natural substrate, a ligand, a structural or functional mimetic. (See,Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which G-protein Chemokine Receptor (CCR5) binds, or at least, afragment of the receptor capable of being bound by G-protein ChemokineReceptor (CCR5) (e.g., active site). In either case, the molecule can berationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express G-protein Chemokine Receptor (CCR5),either as a secreted protein or on the cell membrane. Preferred cellsinclude cells from mammals, yeast, Drosophila, or E. coli. Cellsexpressing G-protein Chemokine Receptor (CCR5) (or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either G-protein ChemokineReceptor (CCR5) or the molecule.

The assay may simply test binding of a candidate compound to G-proteinChemokine Receptor (CCR5), wherein binding is detected by a label, or inan assay involving competition with a labeled competitor. Further, theassay may test whether the candidate compound results in a signalgenerated by binding to G-protein Chemokine Receptor.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining G-protein Chemokine Receptor (CCR5), measuring G-proteinChemokine Receptor/molecule activity or binding, and comparing theG-protein Chemokine Receptor/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure G-protein Chemokine Receptor(CCR5) level or activity in a sample (e.g., biological sample) using amonoclonal or polyclonal antibody. The antibody can measure G-proteinChemokine Receptor (CCR5) level or activity by either binding, directlyor indirectly, to G-protein Chemokine Receptor (CCR5) or by competingwith G-protein Chemokine Receptor (CCR5) for a substrate.

Additionally, the ligands to which G-protein Chemokine Receptor (CCR5)binds can be identified by numerous methods known to those of skill inthe art, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of G-proteinChemokine Receptor (CCR5) thereby effectively generating agonists andantagonists of G-protein Chemokine Receptor. See generally, U.S. Pat.Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, andPatten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997);Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., etal., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference). In one embodiment,alteration of G-protein Chemokine Receptor (CCR5) polynucleotides andcorresponding polypeptides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments into adesired G-protein Chemokine Receptor (CCR5) molecule by homologous, orsite-specific, recombination. In another embodiment, G-protein ChemokineReceptor (CCR5) polynucleotides and corresponding polypeptides may bealtered by being subjected to random mutagenesis by error-prone PCR,random nucleotide insertion or other methods prior to recombination. Inanother embodiment, one or more components, motifs, sections, parts,domains, fragments, etc., of G-protein Chemokine Receptor (CCR5) may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules are G-proteinChemokine Receptor (CCR5) family members. In further preferredembodiments, the heterologous molecule is a growth factor such as, forexample, platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growthfactor (EGF), fibroblast growth factor (FGF), TGF-beta, bonemorphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins Aand B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiationfactors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2,TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active G-protein ChemokineReceptor (CCR5) fragments. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the G-protein Chemokine Receptor (CCR5) polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the G-protein Chemokine Receptor (CCR5) is measured and theability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems includebut are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind toG-protein Chemokine Receptor (CCR5) comprising the steps of: (a)incubating a candidate binding compound with G-protein ChemokineReceptor; and (b) determining if binding has occurred. Moreover, theinvention includes a method of identifying agonists/antagonistscomprising the steps of: (a) incubating a candidate compound withG-protein Chemokine Receptor (CCR5), (b) assaying a biological activity,and (b) determining if a biological activity of G-protein ChemokineReceptor (CCR5) has been altered.

Also, one could identify molecules bind G-protein Chemokine Receptor(CCR5) experimentally by using the beta-pleated sheet regions disclosedin FIG, 3 and Table 1. Accordingly, specific embodiments of theinvention are directed to polynucleotides encoding polypeptides whichcomprise, or alternatively consist of, the amino acid sequence of eachbeta pleated sheet regions disclosed in FIG. 3/Table 1. Additionalembodiments of the invention are directed to polynucleotides encodingG-protein Chemokine Receptor (CCR5) polypeptides which comprise, oralternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 3/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the G-protein Chemokine Receptor(CCR5) amino acid sequence of each of the beta pleated sheet regionsdisclosed in FIG. 3/Table 1. Additional embodiments of the invention aredirected to G-protein Chemokine Receptor (CCR5) polypeptides whichcomprise, or alternatively consist of, any combination or all of thebeta pleated sheet regions disclosed in FIG. 3/Table 1.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNase, alpha toxin, ricin, abrin, Pseudomontas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubicin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Thus, the polypeptides can be used to identify compounds that modulatereceptor activity. Both the receptor protein and appropriate variantsand fragments can be used in high throughput screens to assay candidatecompounds for the ability to bind to the receptor. These compounds canbe further screened against a functional receptor to determine theeffect of the compound on the receptor activity. Compounds can beidentified that activate (agonist) or inactivate (antagonist) thereceptor to a desired degree.

The terms “agonist” and “antagonist” represent compounds that enhance ordiminish a response. As one form of an agonist, the compound binds tothe same site as the endogenous compound and produces the same type ofsignal, usually of equal or greater magnitude than the endogenous agent.Another form of agonist binds to a different site than the firstagonist, producing no signal by itself, however, an enhanced signal isgenerated when the endogenous agent also binds to its site. This iscalled an allosteric action. One form of antagonist binds to the siteused by the endogenous agent and diminishes or blocks the signalgenerated by the endogenous agent. Another form of antagonist binds toan allosteric site, similar to the second form of agonist, but producesa diminished signal generated by the endogenous agent. A third form ofantagonist dissolves in the membrane or crosses the membrane andintercepts the signal generated by the endogenous agent within themembrane or on the intracellular side. An antagonist, accordingly,encompasses negative agonists or “inverse agonists”, having a negativeintrinsic activity that reduces the receptor signal activity relative tothe signaling activity measured in the absence of the inverse agonist.Such an antagonist is distinguished from an antagonist having nointrinsic activity and no effect on the receptor's basal activity. Thus,for example, an inverse agonist could alter the receptor confirmation,thereby reducing or eliminating interaction with a ligand. See, Milliganet al., TIPS 16:10 (1995).

The receptor polypeptides can be used to screen a compound for theability to stimulate or inhibit interaction between the receptor proteinand a target molecule that normally interacts with the receptor protein.The target can be ligand or a component of the signal pathway with whichthe receptor protein normally interacts (for example, a G-protein orother interactor involved in cAMP or phosphatidylinositol turnoverand/or adenylate cyclase, or phospholipase C activation). The assayincludes the steps of combining the receptor protein with a candidatecompound under conditions that allow the receptor protein or fragment tointeract with the target molecule, and to detect the formation of acomplex between the protein and the target or to detect the biochemicalconsequence of the interaction with the receptor protein and the target,such as any of the associated effects of signal transduction, such asion flux, G-protein phosphorylation, cyclic AMP or phosphatidylinositolturnover, and adenylate cyclase or phospholipase C activation.

The receptor polypeptides are useful in cell based assays when they areoverexpressed in a cell. Accordingly, such cells overexpressing thereceptor are useful to identify compounds that are capable of modulatingor compensating for the overexpression. Cells overexpressing thereceptor can be derived from natural sources or can be created byroutine recombinant methods.

The receptor polypeptides are also useful for screening compounds in acell based assay when constitutively activated on a cell. Such cellsexpressing constitutively activated receptors are useful for screeningcompounds that modulate receptor activation. Such cells can be derivedfrom natural sources or can be created by recombinant means that arewell known in the art. For example, see Scheer et al., J. ReceptorSignal Transduction Res. 17:57-73 (1997); U.S. Pat. No. 5,750,353.

Candidate compounds include, for example, (1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; (2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); (3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, intrabodies, and singlechain antibodies, as well as Fab, F(ab)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and (4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

One candidate compound is a soluble full-length receptor or fragmentthat competes for ligand binding. Other candidate compounds includemutant receptors or appropriate fragments containing mutations thataffect receptor function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

The invention provides other end points to identify compounds thatmodulate (stimulate or inhibit) receptor activity. The assays typicallyinvolve an assay of events in the signal transduction pathway thatindicate receptor activity. Thus, the expression of genes that are up-or down-regulated in response to the receptor protein dependent signalcascade can be assayed. In one embodiment, the regulatory region of suchgenes can be operably linked to a marker that is easily detectable, suchas luciferase. Alternatively, phosphorylation of the receptor protein,or a receptor protein target, could also be measured.

It is also understood that a disorder caused by aberrant levels ormutations in the protein can be used as a basis for an endpoint.Accordingly, specific deviations in the development or course of thedisorder in response to a compound that acts on the receptor can serveas an endpoint.

Any of the biological or biochemical functions mediated by the receptorcan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in theart.

Binding and/or activating compounds can also be screened by usingchimeric receptor proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops, and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a G-protein-binding region can beused that interacts with a different G-protein than that which isrecognized by the native receptor. Accordingly, a different set ofsignal transduction components is available as an end-point assay foractivation. Alternatively, the entire transmembrane portion orsubregions (such as transmembrane segments or intracellular orextracellular loops) can be replaced with the entire transmembraneportion or subregions specific to a host cell that is different from thehost cell from which the amino terminal extracellular domain and/or theG-protein binding region are derived. This allows for assays to beperformed in other than the specific host cell from which the receptoris derived. Alternatively, the amino terminal extracellular domain(and/or other ligand-binding regions) could be replaced by a domain(and/or other binding region) binding a different ligand, thus,providing an assay for test compounds that interact with theheterologous amino terminal extracellular domain (or region) but stillcause signal transduction. Finally, activation can be detected by areporter gene containing an easily detectable coding region operablylinked to a transcriptional regulatory sequence that is part of thenative signal transduction pathway.

The receptor polypeptides are also useful in competition binding assaysin methods designed to discover compounds that interact with thereceptor. Thus, a compound is exposed to a receptor polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble receptor polypeptide is also added to themixture. If the test compound interacts with the soluble receptorpolypeptide, it decreases the amount of complex formed or activity fromthe receptor target. This type of assay is particularly useful in casesin which compounds are sought that interact with specific regions of thereceptor. Thus, the soluble polypeptide that competes with the targetreceptor region is designed to contain peptide sequences correspondingto the target region.

To perform cell-free drug screening assays, it is desirable toimmobilize either the receptor protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing G-proteins on matrices can be used in thedrug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-Stransferase/G-protein ChemokineReceptor (CCR5) fusion proteins can be adsorbed onto glutathionesepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathionederivatized microtitre plates, which are then combined with the celllysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofreceptor-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques, For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a receptor-bindingprotein and a candidate compound are incubated in the receptorprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the receptorprotein target molecule, or which are reactive with receptor protein andcompete with the target molecule; as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetmolecule.

Modulators of receptor protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the receptor pathway, by treating cells that express thereceptor protein. These methods of treatment include the steps ofadministering the modulators of protein activity in a pharmaceuticalcomposition as described herein, to a subject in need of such treatment.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 97183. In one embodiment,antisense sequence is generated internally, by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl₂, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the G-protein Chemokine Receptor (CCR5) antisensenucleic acid of the invention is produced intracellularly bytranscription from an exogenous sequence. For example, a vector or aportion thereof, is transcribed, producing an antisense nucleic acid(RNA) of the invention. Such a vector would contain a sequence encodingthe G-protein Chemokine Receptor (CCR5) antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding G-protein Chemokine Receptor (CCR5),or fragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinster,et al., Nature 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a G-proteinChemokine Receptor (CCR5) gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded G-protein ChemokineReceptor (CCR5) antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the larger thehybridizing nucleic acid, the more base mismatches with a G-proteinChemokine Receptor (CCR5) RNA it may contain and still form a stableduplex (or triplex as the case may be). One skilled in the art canascertain a tolerable degree of mismatch by use of standard proceduresto determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′- non-translated, non-coding regions of G-protein Chemokine Receptor(CCR5) shown in FIGS. 1A-B or within the coding region of the depositedclone could be used in an antisense approach to inhibit translation ofendogenous G-protein Chemokine Receptor (CCR5) mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention. Whetherdesigned to hybridize to the 5′-, 3′- or coding region of G-proteinChemokine Receptor (CCR5) mRNA or within the coding region of thedeposited clone, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the G-protein ChemokineReceptor (CCR5) coding region sequence could be used, thosecomplementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy G-protein Chemokine Receptor (CCR5)mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach, Nature 334:585-591 (1988). There are numerous potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofG-protein Chemokine Receptor (CCR5) (FIGS. 1A-B or the sequence of thedeposited clone). Preferably, the ribozyme is engineered so that thecleavage recognition site is located near the St end of the G-proteinChemokine Receptor (CCR5) mRNA; i.e., to increase efficiency andminimize the intracellular accumulation of non-functional mRNAtranscripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which expressG-protein Chemokine Receptor (CCR5) in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous G-proteinChemokine Receptor (CCR5) messages and inhibit translation. Sinceribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Use of Transmembrane Fragments as Antagonists/Inhibitors

In specific embodiments, the present invention relates to modulating,especially inhibiting, biological activities of G-protein ChemokineReceptor (CCR5) by exposing it to molecules which interfere with correctreceptor assembly. In particular, the invention relates to isolatedfragments or peptides of the transmembrane domain of G-protein ChemokineReceptor (CCR5) that inhibit G-protein Chemokine Receptor mediatedsignal transduction or ligand-binding. In certain embodiments, chargedresidues are added at one terminus of the fragment to promote correctorientation of the fragment in the membrane.

It has been demonstrated that G-protein coupled receptor (GPCR)transmembrane (TM) domain segments interact in a specific way during theassembly of receptor molecules. These interactions do not lead to arigid structure because some flexibility is required for conformationalchanges following ligand binding, which allow the molecule to signalfrom the cell surface to the intracellular parts. It was alsodemonstrated for several GPCRs that the transmembrane domain is involvedin ligand binding and thus it contains openings that allow penetrationof the ligands. Reports that expression of missing transmembrane domainsrescues inactive truncated V2 vasopressin, beta-adrenergic andmuscarinic M3 receptors (Schoneberg et al. EMBO J. 15:1283 (1996); Wonget al., J. Biol. Chem. 265:6219 (1990); Monnot et al., J. Biol. Chem.271:1507 (1996); Gudermann et al., Annu. Rev. Neurosci. 20:399 (1997);Osuga et al., J. Biol. Chem. 272:25006 (1997)) suggested peptide derivedfrom the sixth transmembrane domain of P2-adrenergic receptor was foundto inhibit receptor activation and dimerization (Hebert et al., J. Biol.Chem., 271(27):16384-92 (1996)). Thus, GPCR function can be rescued bytargeting intramembrane interactions of GPCRs.

Additionally, GPCR function can be inhibited by targeting intramembraneinteractions. For example, fragments corresponding to predicted TMsegments of CCR5, which functions as a coreceptor during entry of HIVinto cells, yielded potent inhibition of HIV entry without apparenttoxicity to the cells. (See WO 99/43711) The usefulness of the methodwas also demonstrated by specifically targeting CXCR4, which functionsas a co-receptor during the cell entry of T-cell tropic strains ofHIV-1. Fragments containing 20-25 amino acid residues inhibited receptorsignaling and HIV-1 infection in vitro at concentration as low as 0.2micromolar. (See WO 99/43711).

The hydrophobic and/or amphipathic nature of the transmembrane fragmentsallows their penetration into the bilayer. Further, orientation insidethe membrane can be controlled by addition of charged residues to theterminus that is exposed at the extracellular side of the membrane inthe intact receptor. Insertion into the membrane is tested byfluorescent microscopy of labeled peptide analogs using methodologyknown to those of ordinary skill in the art, and as described in WO99/43711.

In a particular embodiment, the invention encompasses transmembranefragments that modulate, and preferably inhibit the biologicalproperties and activities of G-protein Chemokine Receptor (CCR5), bytargeting the transmembrane domain of this receptor. In a furtherembodiment, the invention specifically comprises methods for disruptingG-protein Chemokine Receptor (CCR5) function by using these antagonists.

Chemical or recombinant DNA technology may be used to obtain G-proteinChemokine Receptor (CCR5) transmembrane fragments, which preferably areas small as possible while still retaining sufficiently high affinityfor binding to, or association with, G-protein Chemokine Receptor.Non-limiting examples of G-protein Chemokine Receptor (CCR5)polypeptides include fragments of 10 to 50 amino acids corresponding toat least one transmembrane segment of segments 1-7.

In another embodiment, the invention encompasses an isolated G-proteinChemokine Receptor-modulating molecule comprising a fragment, a peptide,or peptidomimetic that is a structural analog of a portion of atransmembrane segment of G-protein Chemokine Receptor (CCR5), whereinsaid molecule has an extracellular end and an intracellular end and saidmolecule has at said extracellular end a negatively charged group and atsaid intracellular end a neutral charge under physiological conditions;said molecule spontaneously inserts into a membrane in the sameorientation as the transmembrane domain from which it is derived; andsaid molecule modulates a biological property or activity of saidG-protein Chemokine Receptor.

In a particular embodiment, the molecules contain a hydrophilic,negatively charged non-peptidic head group and an uncharged tail, whichassures correct orientation of the molecule in the cell membrane. Inanother embodiment, the negatively charged head group is one or moreacidic amino acids.

The G-protein Chemokine Receptor (CCR5) activity modulated by saidfragment includes inhibition of G-protein Chemokine Receptor-mediatedintracellular Ca²⁺ release and inhibition of G-protein ChemokineReceptor-mediated HIV infection. The G-protein Chemokine Receptor (CCR5)activity modulated by said peptide also includes binding of a G-proteinChemokine Receptor (CCR5) ligand. Additional G-protein ChemokineReceptor (CCR5) activities modulated by said peptide include those inthe Description and Examples herein.

In another embodiment, the invention comprises methods of modulating thebiological activity of a G-protein Chemokine Receptor (CCR5) bycontacting a cell that expresses G-protein Chemokine Receptor (CCR5)with a molecule of the invention. In one method, the modulatedbiological activity is inhibition of G-protein ChemokineReceptor-mediated HIV infection. In another method, the modulatedbiological activity is inhibition of G-protein ChemokineReceptor-mediated intracellular Ca²⁺ release. Other activities modulatedinclude those described elsewhere herein.

Another embodiment is a method of inhibiting HIV-1 infection, comprisingcontacting a cell that expresses G-protein Chemokine Receptor (CCR5)which binds HIV-1 with a molecule that comprises a polypeptide fragment,a peptide, or peptidomimetic that is a structural analog of a portion ofthe transmembrane domain of said G-protein Chemokine Receptor (CCR5),wherein contacting the cell with said molecule inhibits HIV-1 infection.The peptide or peptidomimetic may be a structural analog of a portion ofa transmembrane domain of G-protein Chemokine Receptor.

In one embodiment, the molecules of the present invention mimic atransmembrane segment of G-protein Chemokine Receptor (CCR5) and blockself-assembly of the receptor, possibly by competitive inhibition withthe native TM segment. They thereby block or inhibit signal transductionin the affected cell.

The invention also includes peptide analogs and peptidomimetics whichpossess beneficial properties such as increased half-life, lack ofimmunogenicity, and the ability to cross the blood-brain barrier.

The peptide analogs of the invention mediate the chemical and/orbiological effects of hormone agonists/antagonists or other peptides.They are useful for the development of pharmaceutical, therapeutic, anddiagnostic techniques. Accordingly, the invention also provides methodsfor producing a prophylactic or therapeutic response in a mammal byadministering to the mammal a pharmaceutically effective amount of oneor more peptide analogs of the invention. In preferred embodiments, thepresent invention provides methods for producing such responses bymodulating the activity of G-protein Chemokine Receptor (CCR5) byadministering an effective amount of one or more peptide analogs of theinvention.

In another embodiment, more than one peptide of the invention areadministered as a cocktail to modulate the biological activity ofG-protein Chemokine Receptor.

The term “G-protein Chemokine Receptor (CCR5) transmembrane peptide” caninclude a fragment of the transmembrane domain, a transmembrane segment,a fragment of a transmembrane segment, and/or a homologous peptidethereof. Preferred fragments include those of at least 4-50, andpreferably at least 4-30, and preferably at least 10-30 amino acids inlength, or any range therein. Further preferred fragments include thoseof 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Alsoincluded are any corresponding sequences having conservative amino acidsubstitutions.

Sample transmembrane fragments of the invention include, but are notlimited to, any fragment described herein. A preferred transmembranefragment of the present invention, when contacted with a cell ormembrane structure (e.g., liposome) that contains biologically activeG-protein Chemokine Receptor (CCR5), modulates the biological activityof said G-protein Chemokine Receptor (CCR5) in vitro, in vivo, or insitu. The concentration of the fragment in a solution that contacts thecell in vivo (e.g., blood plasma or interstitial fluid) or in vitro(e.g., culture medium) is between 1 nanomolar and 50 micromolar,preferably between 1 nanomolar and 1 micromolar, and most preferablyless than 5 micromolar.

“Negatively charged” refers to those amino acids, amino acidderivatives, amino acid mimetics and chemical moieties that arenegatively charged at physiological pH. Negatively charged amino acidsinclude, for example Asp and Glu. An “acidic” residue is a residue thatis negatively charged at physiological pH.

“Positively charged” refers to those amino acids, amino acidderivatives, amino acid mimetics and chemical moieties that arepositively charged at physiological pH. Positively charged amino acidsinclude, for example, Lys and Arg. A “basic residue” is a residue thatis positively charged at physiological pH.

“Neutral” refers to those amino acids, amino acid derivatives, aminoacid mimetics and chemical moieties that are neither positively nornegatively charged at physiological pH.

The term “modulates a biological property or activity” means that in thepresence of a test transmembrane fragment a measurable biologicalparameter or event is increased or decreased relative to a control inthe absence of said peptide. Examples of biological property or activityinclude: conformation of G-protein Chemokine Receptor (CCR5),association of the G-protein Chemokine Receptor (CCR5) with othermolecules, signal transduction, extracellular secretion of cellularproteins, conformational changes in proteins, changes in enzymaticactivity, changes in metabolic activity, changes in affinity for aligand, changes in levels of viral infection, changes in vasodilation,modulation of heart rate, modulation of bronchodilation, modulation ofendocrine secretions, and modulation of gut peristalsis. Note that theG-protein Chemokine Receptor (CCR5) biological activity need not be onethat is limited to the precise in vivo role performed by the G-proteinChemokine Receptor. The term also covers G-protein Chemokine Receptor(CCR5) properties, such as viral protein binding, that are not part ofthe in vivo biological role of the G-protein Chemokine Receptor. Itfurther covers intrinsic properties of G-protein Chemokine Receptor(CCR5) that are only disclosed by experimental manipulation in thelaboratory, such as the ability of G-protein Chemokine Receptor (CCR5)in artificial bilayers (e.g., liposomes) to interact with G-proteinChemokine Receptor (CCR5) ligands.

“Signal transduction” is the process by which binding of a ligand to areceptor is translated into physiological change. In general, binding ofa ligand to a receptor causes a change in a physical property of thereceptor, for example a change in its conformation, or its orientation,or in its ability to bind other ligands. This change in a physicalproperty can result, directly or indirectly, in increased or decreasedion fluxes, increased or decreased enzymatic activity, increased ordecreased phosphorylation, increased or decreased translocation of thereceptor or of any molecule (e.g., an inositol moiety or a G-proteinsubunit) from one cellular compartment to another.

“G-protein Chemokine Receptor (CCR5) ligands” refers to biologicalmolecules that bind G-protein Chemokine Receptor (CCR5) in vitro, insitu, or in vivo, and may include hormones, neurotransmitters, virusesor receptor-binding domains thereof, G-proteins, opsins, rhodopsins,nucleosides, nucleotides, coagulation cascade factors, odorants orpheromones, toxins, colony stimulating factors, platelet activatingfactors, neuroactive peptides, neurohumor, or any biologically activecompounds, such as drugs or synthetic or naturally occurring compounds.

The phrase “inhibits HIV infection” means that a peptide of theinvention inhibits binding of a HIV to G-protein Chemokine Receptor(CCR5) or inhibits a G-protein Chemokine Receptor (CCR5) biologicalactivity that mediates the entry and successful reproduction of a HIVvirus into a G-protein Chemokine Receptor-expressing cell.

G-protein Chemokine Receptor (CCR5) polypeptides of the presentinvention, or nucleic acids encoding therefor, include a finite set ofsubstantially corresponding sequences as substitution peptides orpolynucleotide which can be routinely obtained by one of ordinary skillin the art, without undue experimentation, based on the teachings andguidance presented herein. For a detailed description of proteinchemistry and structure, see Schulz et al., PRINCIPLES OF PROTEINSTRUCTURE, Springer-Verlag, New York, 1978, and Creighton, T. E.,PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES, W.H. Freeman & Co., SanFrancisco, 1983, which are hereby incorporated by reference. For apresentation of nucleotide sequence substitutions, such as codonpreferences, see Ausubel et al, supra, at sections A.1,1-A.1.24, andSambrook et al., supra, at Appendices C and D.

G-protein Chemokine Receptor (CCR5) polypeptides include homologoussequences and/or fragments of the transmembrane domain, in particular,at least one of transmembrane segment 1-7 of G-protein ChemokineReceptor (CCR5) or homologs thereof.

However, in the context of the present invention, G-protein ChemokineReceptor (CCR5) polypeptides of at least 15-20 amino acids are preferredsuch that the G-protein Chemokine Receptor (CCR5) polypeptides are ableto span the lipid bilayer.

It is particularly preferred that peptides of the invention be selectedor modified so that one end is charged and the other is neutral underphysiological conditions. This is so that the peptide spontaneouslyinserts into a membrane. It is of particular importance that the peptideinsert in the same orientation as the transmembrane G-protein ChemokineReceptor (CCR5) domain from which it is derived.

Peptides of the invention can be derived from any of the 7 TM segments.The amino acid positions of the TM segments can be determined usingmolecular modeling, optionally combined with hydrophobicity analysis(see Table 1), and/or fitting to model helices, as non-limitingexamples. Such modeling can be accomplished according to known methods,for example, ECEPP, INSIGHT, DISCOVER, CHEM-DRAW, AMBER, FRODO, andCHEM-X. Such algorithms compare transmembrane domains and segmentsbetween related GPCRs, determine probable energy-minimized structuresand define alternative transmembrane sequences.

Fragments of the G-protein Coupled Chemokine Receptor transmembranedomain which are useful as antagonists comprise, or alternativelyconsist of, the following portions of SEQ ID NO:2 or of the polypeptideencoded by the deposited clone:

Segment 1: amino acids 31-58, 32-58, 33-58, 34-58, 35-58, 36-58, 37-58,38-58, 31-57, 32-57, 33-57, 34-57, 35-57, 36-57, 37-57, 31-56, 32-56,33-56, 34-56, 35-56, 36-56, 31-55, 32-55, 33-55, 34-55, 35-55, 31-54,32-54, 33-54, 34-54, 31-53, 32-53, 33-53, 31-52, 32-52, 31-51 of SEQ IDNO:2 or of the polypeptide encoded by the deposited clone, or anycombination thereof, preferably at least 15 amino acids in length;

Segment 2: amino acids 68-97, 69-97, 70-97, 71-97, 72-97, 73-97, 74-97,75-97, 76-97, 68-96, 69-96, 70-96, 71-96, 72-96, 73-96, 74-96, 75-96,76-96, 68-95, 69-95, 70-95, 71-95, 72-95, 73-95, 74-95, 75-95, 68-94,69-94, 70-94, 71-94, 72-94, 73-94, 74-94, 68-93, 69-93, 70-93, 71-93,72-93, 73-93, 68-92, 69-92, 70-92, 71-92, 72-92, 68-91, 69-91, 70-91,71-91, 68-90, 69-90, 70-90, 68-89, 69-89, 68-88 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone, or any combination thereof,preferably at least 15 amino acids in length;

Segment 3: amino acids 103-124, 104-124, 105-124, 106-124, 107-124,108-124, 109-124, 103-123, 103-122, 103-121, 103-120, 103-119, 103-118of SEQ ID NO:2 or of the polypeptide encoded by the deposited clone, orany combination thereof, preferably at least 15 amino acids in length;

Segment 4: amino acids 142-169, 143-169, 144-169, 145-169, 146-169,147-169, 148-169, 149-169, 142-168, 143-168, 144-168, 145-168, 146-168,147-168, 148-168, 142-167, 143-167, 144-167, 145-167, 146-167, 147-167,142-166, 143-166, 144-166, 145-166, 146-166, 142-165, 143-165, 144-165,145-165, 142-164, 143-164, 144-164, 142-163, 143-163, 142-163 of SEQ IDNO:2 or of the polypeptide encoded by the deposited clone, or anycombination thereof, preferably at least 15 amino acids in length;

Segment 5: amino acids 196-223, 197-223, 198-223, 199-223, 200-223,201-223, 202-223, 203-223, 196-222, 197-222, 198-222, 199-222, 200-222,201-222, 202-222, 196-221, 197-221, 198-221, 199-221, 200-221, 201-221,196-220, 197-220, 198-220, 199-220, 200-220, 196-219, 197-219, 198-219,199-219, 196-218, 197-218, 198-218, 196-217, 197-217, 196-216 of SEQ IDNO:2 or of the polypeptide encoded by the deposited clone, or anycombination thereof, preferably at least 15 amino acids in length,

Segment 6: amino acids 236-260, 237-260, 238-260, 239-260, 240-260,236-259, 237-259, 238-259, 239-259, 236-258, 237-258, 238-258, 236-257,237-257, 236-256 of SEQ ID NO:2 or of the polypeptide encoded by thedeposited clone, or any combination thereof, preferably at least 15amino acids in length;

Segment 7: amino acids 275-305, 276-305, 277-305, 278-305, 279-305,280-305, 281-305, 282-305, 283-305, 284-305, 285-305, 275-304, 276-304,277-304, 278-304, 279-304, 280-304, 281-304, 282-304, 283-304, 284-304,275-303, 276-303, 277-303, 278-303, 279-303, 280-303, 281-303, 282-303,283-303, 275-302, 276-302, 277-302, 278-302, 279-302, 280-302, 281-302,282-302, 275-301, 276-301, 277-301, 278-301, 279-301, 280-301, 281-301,275-300, 276-300, 277-300, 278-300, 279-300, 280-300, 275-299, 276-299,277-299, 278-299, 279-299, 275-298, 276-298, 277-298, 278-298, 275-297,276-297, 277-297, 275-296, 276-296, 275-295 of SEQ ID NO:2 or of thepolypeptide encoded by the deposited clone, or any combination thereof,preferably at least 15 amino acids in length;

The CCR5 fragments disclosed in WO 99/43711 are specifically excludedfrom the embodiments in this section.

Negatively charged amino acids, such as Asp or Glu, may be substitutedor added at the extracellular end of the fragment. The number ofnegatively charged amino acids is typically 1, 2, or 3. Neutral aminoacids may be substituted or added at the intracellular end of thefragment. See, also, Example 57.

Other Activities

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention, as a result of the ability to stimulate vascular endothelialcell growth, may be employed in treatment for stimulatingre-vascularization of ischemic tissues due to various disease conditionssuch as thrombosis, arteriosclerosis, and other cardiovascularconditions. The polypeptide, polynucleotide, agonist, or antagonist ofthe present invention may also be employed to stimulate angiogenesis andlimb regeneration, as discussed above.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for treating, preventing, and/ordiagnosing wounds due to injuries, burns, post-operative tissue repair,and ulcers since they are mitogenic to various cells of differentorigins, such as fibroblast cells and skeletal muscle cells, andtherefore, facilitate the repair or replacement of damaged or diseasedtissue.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed stimulate neuronal growth and to treatand prevent neuronal damage which occurs in certain neuronal diseases,disorders, and/or conditions or neuro-degenerative conditions such asAlzheimer's disease, Parkinson's disease, and AIDS-related complex. Apolypeptide, polynucleotide, agonist, or antagonist of the presentinvention may have the ability to stimulate chondrocyte growth,therefore, they may be employed to enhance bone and periodontalregeneration and aid in tissue transplants or bone grafts.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be also be employed to prevent skin aging due to sunburnby stimulating keratinocyte growth.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for preventing hair loss, since FGFfamily members activate hair-forming cells and promotes melanocytegrowth. Along the same lines, a polypeptide, polynucleotide, agonist, orantagonist of the present invention may be employed to stimulate growthand differentiation of hematopoietic cells and bone marrow cells whenused in combination with other cytokines.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed to maintain organs before transplantationor for supporting cell culture of primary tissues. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may alsobe employed for inducing tissue of mesodermal origin to differentiate inearly embryos.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used to modulate mammalian characteristics, suchas body height, weight, hair color, eye color, skin, percentage ofadipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, a polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may be used to modulate mammalian metabolism affectingcatabolism, anabolism, processing, utilization, and storage of energy.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be used to change a mammal's mental state or physicalstate by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used as a food additive or preservative, such asto increase or decrease storage capabilities, fat content, lipid,protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Bacterial Expression and Purification of HDGNR10

The DNA sequence encoding for HDGNR10, ATCC No. 97183 is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ andsequences of the processed HDGNR10 protein (minus the signal peptidesequence) and the vector sequences 3′ to the HDGNR10 gene. Additionalnucleotides corresponding to HDGNR10 were added to the 5′ and 3′sequences respectively. The 5′ oligonucleotide primer has the sequence5′ CGGAATTCCTCCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:3) and contains an EcoRIrestriction enzyme site followed by 18 nucleotides of HDGNR10 codingsequence starting from the presumed terminal amino acid of the processedprotein codon.

The 3′ sequence 5′CGGAAGCTTCGTCACAAGCCCACAGATAT 3′ (SEQ ID NO:4)contains complementary sequences to a HindIII site and is followed by 18nucleotides of HDGNR10 coding sequence. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311).pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 was then digested with EcoRI and HindIII. The amplified sequenceswere ligated into pQE-9 and were inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture wasthen used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by theprocedure described in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies were selected. Plasmid DNA wasisolated and confirmed by restriction analysis.

Clones containing the desired constructs were grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 μg/ml) andKan (25 μg/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells were grown to an optical density600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized HDGNR10 was purified from this solution by chromatography ona Nickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography411:177-184 (1984). HDGNR10 was eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

Example 2 Expression of Recombinant HDGNR10 in COS Cells

The expression of plasmid HDGNR10 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire HDGNR10 precursor and a HA tag fused inframe to its 3′ end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, et al., Cell37:767 (1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for HDGNR10, ATCC No. 97183, was constructedby PCR using two primers: the 5′ primer 5′ GTCCAAGCTTGCCACCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:5) and contains a HindIII site followed by 18nucleotides of HDGNR10 coding sequence starting from the initiationcodon; the 3′ sequence 5′ CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCACAAGCCCACAGATATTTC 3′ (SEQ ID NO:6) contains complementary sequences to anXhoI site, translation stop codon, HA tag and the last 18 nucleotides ofthe HDGNR10 coding sequence (not including the stop codon). Therefore,the PCR product contains a HindIII site HDGNR10 coding sequence followedby HA tag fused in frame, a translation termination stop codon next tothe HA tag, and an XhoI site. The PCR amplified DNA fragment and thevector, pcDNAI/Amp, were digested with HindIII and XhoI restrictionenzyme and ligated. The ligation mixture was transformed into E. colistrain SURE (available from Stratagene Cloning Systems, 11099 NorthTorrey Pines Road, La Jolla, Calif. 92037) the transformed culture wasplated on ampicillin media plates and resistant colonies were selected.Plasmid DNA was isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment. For expression of therecombinant HDGNR10, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the HDGNR10 HA protein was detected byradiolabelling and immunoprecipitation method. (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)Both cell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

Example 3 Cloning and Expression of HDGNR10 Using the BaculovirusExpression System

The DNA sequence encoding the full length HDGNR10 protein, ATCC No.97183, was amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence

5′CGGGATCCCTCCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:7) and contains a BamHIrestriction enzyme site followed by 4 nucleotides resembling anefficient signal for the initiation of translation in eukaryotic cells(Kozak, M., J. Mol. Biol. 196:947-950 (1987)) and just behind the first18 nucleotides of the HDGNR10 gene (the initiation codon for translationis “ATG”).

The 3′ primer has the sequence

5′CGGGATCCCGCTCACAAGCCCACAGATAT 3′ (SEQ ID NO:8) and contains thecleavage site for the restriction endonuclease BamHI and 18 nucleotidescomplementary to the 3′ non-translated sequence of the HDGNR10 gene. Theamplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamHI andpurified as described above. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the HDGNR10 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonuclease BamHI. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant viruses thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofco-transfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology 170:31-39).

The plasmid was digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA was then isolated from a 1% agarose gel as describedabove. This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBacHDGNR10) with the HDGNR10gene using the enzyme BamHI. The sequence of the cloned fragment wasconfirmed by DNA sequencing.

5 μg of the plasmid pBacHDGNR10 were co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner, et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacHDGNR10 weremixed in a sterile well of a microtiter plate containing 50 μg of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, pages 9-10).

Four days after the serial dilution, the viruses were added to thecells, blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculoviruses was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-HDGNR10 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Example 4 Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added). This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA 7:219-25 (1988) flanked by the longterminal repeats of the Moloney murine sarcoma virus, is digested withEcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer contains an EcoRI site and the 3′ primercontains a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the EcoRI and HindIII fragment are addedtogether, in the presence of T4 DNA ligase. The resulting mixture ismaintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+aml2 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles medium (DMEM)with 10% calf serum (CS) penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 5 Isolation of the G-Protein Chemokine Receptor (CCR5) DNA Clonefrom the Deposited Sample

The DNA for G-protein Chemokine Receptor (CCR5) is inserted into themultiple cloning site of pQE-9. (Qiagen, Inc.) pQE-9 contains anAmpicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Gruber, C.E., et al., Focus 15:59-(1993).)

Two approaches can be used to isolate G-protein Chemokine Receptor(CCR5) from the deposited sample. First, the deposited clone istransformed into a suitable host (such as XL-1 Blue (Stratagene)) usingtechniques known to those of skill in the art, such as those provided bythe vector supplier or in related publications or patents. Thetransformants are plated on 1.5% agar plates (containing the appropriateselection agent, e.g., ampicillin) to a density of about 150transformants (colonies) per plate. A single colony is then used togenerate DNA using nucleic acid isolation techniques well known to thoseskilled in the art. (e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edit., (1989), Cold Spring Harbor LaboratoryPress.)

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:1 or SEQ ID NO:21 (i.e., within the region of SEQ IDNO:1 or SEQ ID NO:21 bounded by the 5′ NT and the 3′ NT of the clone)are synthesized and used to amplify the G-protein Chemokine Receptor(CCR5) DNA using the deposited DNA plasmid as a template. The polymerasechain reaction is carried out under routine conditions, for instance, in25 ul of reaction mixture with 0.5 ug of the above DNA template. Aconvenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20uM each of dATP, dCTP, dGTP, dTTP, 25 μmol of each primer and 0.25 Unitof Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degreeC. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72degree C. for 1 min) are performed with a Perkin-Elmer Cetus automatedthermal cycler. The amplified product is analyzed by agarose gelelectrophoresis and the DNA band with expected molecular weight isexcised and purified. The PCR product is verified to be the selectedsequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of the G-protein Chemokine Receptor (CCR5) genewhich may not be present in the deposited clone. These methods includebut are not limited to, filter probing, clone enrichment using specificprobes, and protocols similar or identical to 5′ and 3′ “RACE” protocolswhich are well known in the art. For instance, a method similar to 5′RACE is available for generating the missing 5′ end of a desiredfull-length transcript. (Fromont-Racine et al., Nucleic Acids Res.21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of theG-protein Chemokine Receptor (CCR5) gene of interest is used to PCRamplify the 5′ portion of the G-protein Chemokine Receptor (CCR5)full-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the G-protein ChemokineReceptor (CCR5) gene.

Example 6 Isolation of G-protein Chemokine Receptor (CCR5) GenomicClones

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the DNA sequence corresponding to SEQ ID NO:1or SEQ ID NO:21, according to the method described in Example 5. (SeeAlso, Sambrook.)

Example 7 Tissue Distribution of G-protein Chemokine Receptor (CCR5)Polypeptides

Tissue distribution of mRNA expression of G-protein Chemokine Receptor(CCR5) is determined using protocols for Northern blot analysis,described by, among others, Sambrook et al. For example, a G-proteinChemokine Receptor (CCR5) probe produced by the method described inExample 5 is labeled with P³² using the rediprime™ DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe is purified using CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70 degree C. overnight, and the films developed according to standardprocedures.

Example 8 Chromosomal Mapping of G-Protein Chemokine Receptor

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:1 or SEQ ID NO:21. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds, 95degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle isrepeated 32 times followed by one 5 minute cycle at 70 degree C. Human,mouse, and hamster DNA is used as template in addition to a somatic cellhybrid panel containing individual chromosomes or chromosome fragments(Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gelsor 3.5% agarose gels. Chromosome mapping is determined by the presenceof an approximately 100 bp PCR fragment in the particular somatic cellhybrid.

Example 9 Bacterial Expression of G-Protein Chemokine Receptor

G-protein Chemokine Receptor (CCR5) polynucleotide encoding a G-proteinChemokine Receptor (CCR5) polypeptide invention is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ ends of the DNAsequence, as outlined in Example 5, to synthesize insertion fragments.The primers used to amplify the cDNA insert should preferably containrestriction sites, such as BamHI and XbaI, at the 5′ end of the primersin order to clone the amplified product into the expression vector. Forexample, BamHI and XbaI correspond to the restriction enzyme sites onthe bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth,Calif.). This plasmid vector encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatablepromoter/operator (P/O), a ribosome binding site (RBS), a 6-histidinetag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified G-protein Chemokine Receptor (CCR5) protein is thenrenatured by dialyzing it against phosphate-buffered saline (PBS) or 50mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, theG-protein Chemokine Receptor (CCR5) protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedG-protein Chemokine Receptor (CCR5) protein is stored at 4 degree C. orfrozen at −80 degree C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a G-protein Chemokine Receptor(CCR5) polynucleotide, called pHE4a. (ATCC Accession Number 209645,deposited Feb. 25, 1998.) This vector contains: 1) aneomycinphosphotransferase gene as a selection marker, 2) an E. coliorigin of replication, 3) a T5 phage promoter sequence, 4) two lacoperator sequences, 5) a Shine-Delgarno sequence, and 6) the lactoseoperon repressor gene (lacIq). The origin of replication (oriC) isderived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence andoperator sequences are made synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 5, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 10 Purification of G-protein Chemokine Receptor (CCR5)Polypeptide from an Inclusion Body

The following alternative method can be used to purify G-proteinChemokine Receptor (CCR5) polypeptide expressed in E. coli when it ispresent in the form of inclusion bodies. Unless otherwise specified, allof the following steps are conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the G-protein Chemokine Receptor (CCR5) polypeptideare then pooled and mixed with 4 volumes of water. The diluted sample isthen loaded onto a previously prepared set of tandem columns of stronganion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20,Perseptive Biosystems) exchange resins. The columns are equilibratedwith 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mMsodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then elutedusing a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mMsodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5.Fractions are collected under constant A₂₈₀ monitoring of the effluent.Fractions containing the polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant G-protein Chemokine Receptor (CCR5) polypeptide shouldexhibit greater than 95% purity after the above refolding andpurification steps. No major contaminant bands should be observed fromCommassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein isloaded. The purified G-protein Chemokine Receptor (CCR5) protein canalso be tested for endotoxin/LPS contamination, and typically the LPScontent is less than 0.1 ng/ml according to LAL assays.

Example 11 Cloning and Expression of G-Protein Chemokine Receptor (CCR5)in a Baculovirus Expression System

In this example, the plasmid shuttle vector pA2 is used to insertG-protein Chemokine Receptor (CCR5) polynucleotide into a baculovirus toexpress G-protein Chemokine Receptor. This expression vector containsthe strong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sitessuch as BamHI, Xba I and Asp718. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedG-protein Chemokine Receptor (CCR5) polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the G-protein Chemokine Receptor (CCR5) cDNA sequencecontained in the deposited clone, including the AUG initiation codon andany naturally associated leader sequence, is amplified using the PCRprotocol described in Example 5. If the naturally occurring signalsequence is used to produce the secreted protein, the pA2 vector doesnot need a second signal peptide. Alternatively, the vector can bemodified (pA2 GP) to include a baculovirus leader sequence, using thestandard methods described in Summers et al., “A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transfectedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGoId™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced G-protein Chemokine Receptor (CCR5) protein.

Example 12 Expression of G-Protein Chemokine Receptor (CCR5) inMammalian Cells

G-protein Chemokine Receptor (CCR5) polypeptide can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HUVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2DHFR (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, G-protein Chemokine Receptor (CCR5) polypeptide can beexpressed in stable cell lines containing the G-protein ChemokineReceptor (CCR5) polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as DHFR, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected G-protein Chemokine Receptor (CCR5) gene can also beamplified to express large amounts of the encoded protein. The DHFR(dihydrofolate reductase) marker is useful in developing cell lines thatcarry several hundred or even several thousand copies of the gene ofinterest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370(1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta,1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology9:64-68 (1991).) Another useful selection marker is the enzyme glutaminesynthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbingtonet al., Bio/Technology 10:169-175 (1992). Using these markers, themammalian cells are grown in selective medium and the cells with thehighest resistance are selected. These cell lines contain the amplifiedgene(s) integrated into a chromosome. Chinese hamster ovary (CHO) andNSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No. 209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofG-protein Chemokine Receptor. The vectors also contain the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene, and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6 or pC4 is digested with restrictionenzymes that cut within in the multiple cloning site and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The vector can be modified to include a heterologous signal sequence inan effort to secrete the protein from the cell. (See, e.g., WO96/34891.)

The amplified fragment is then digested with restriction enzymes thatgenerate ends complementary to those of the digested vector and purifiedon a 1% agarose gel using a commercially available kit (“Geneclean,” BIO101 Inc., La Jolla, Calif.). The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 or pC4using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHF gene is used fortransfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of methothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of G-protein Chemokine Receptor(CCR5) is analyzed, for instance, by SDS-PAGE and Western blot or byreversed phase HPLC analysis.

Example 13 Construction of N-Terminal and/or C-Terminal Deletion Mutants

The following general approach may be used to clone a N-terminal orC-terminal deletion G-protein Chemokine Receptor (CCR5) deletion mutant.Generally, two oligonucleotide primers of about 15-25 nucleotides arederived from the desired 5′ and 3′ positions of a polynucleotide of SEQID NO:1 or of the deposited clone (SEQ ID NO:21). The 5′ and 3′positions of the primers are determined based on the desired G-proteinChemokine Receptor (CCR5) polynucleotide fragment. An initiation andstop codon are added to the 5′ and 3′ primers respectively, ifnecessary, to express the G-protein Chemokine Receptor (CCR5)polypeptide fragment encoded by the polynucleotide fragment. PreferredG-protein Chemokine Receptor (CCR5) polynucleotide fragments are thoseencoding the N-terminal and C-terminal deletion mutants disclosed abovein the “Polynucleotide and Polypeptide Fragments” section of theSpecification.

Additional nucleotides containing restriction sites to facilitatecloning of the G-protein Chemokine Receptor (CCR5) polynucleotidefragment in a desired vector may also be added to the 5′ and 3′ primersequences. The G-protein Chemokine Receptor (CCR5) polynucleotidefragment is amplified from genomic DNA or from the deposited cDNA cloneusing the appropriate PCR oligonucleotide primers and conditionsdiscussed herein or known in the art. The G-protein Chemokine Receptor(CCR5) polypeptide fragments encoded by the G-protein Chemokine Receptor(CCR5) polynucleotide fragments of the present invention may beexpressed and purified in the same general manner as the full lengthpolypeptides, although routine modifications may be necessary due to thedifferences in chemical and physical properties between a particularfragment and full length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the G-protein Chemokine Receptor (CCR5)polypeptide fragment Y-37 to Q-280 is amplified and cloned as follows: A5′ primer is generated comprising a restriction enzyme site followed byan initiation codon in frame with the polynucleotide sequence encodingthe N-terminal portion of the polypeptide fragment beginning with Y-37.A complementary 3′ primer is generated comprising a restriction enzymesite followed by a stop codon in frame with the polynucleotide sequenceencoding C-terminal portion of the G-protein Chemokine Receptor (CCR5)polypeptide fragment ending with Q-280.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. TheG-protein Chemokine Receptor (CCR5) polynucleotide fragment is insertedinto the restricted expression vector, preferably in a manner whichplaces the G-protein Chemokine Receptor (CCR5) polypeptide fragmentcoding region downstream from the promoter. The ligation mixture istransformed into competent E. coli cells using standard procedures andas described in the Examples herein. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Example 14 Protein Fusions of G-Protein Chemokine Receptor

G-protein Chemokine Receptor (CCR5) polypeptides are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of G-protein Chemokine Receptor (CCR5)polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltosebinding protein facilitates purification. (See Example 9; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to G-protein Chemokine Receptor(CCR5) polypeptides can target the protein to a specific subcellularlocalization, while covalent heterodimer or homodimers can increase ordecrease the activity of a fusion protein. Fusion proteins can alsocreate chimeric molecules having more than one function. Finally, fusionproteins can increase solubility and/or stability of the fused proteincompared to the non-fused protein. All of the types of fusion proteinsdescribed above can be made by modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule, or the protocoldescribed in Example 9.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and G-proteinChemokine Receptor (CCR5) polynucleotide, isolated by the PCR protocoldescribed in Example 5, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal-peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region:

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO:10)

Example 15 Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing G-protein Chemokine Receptor (CCR5) areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of G-proteinChemokine Receptor (CCR5) protein is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

Monoclonal antibodies specific for G-protein Chemokine Receptor (CCR5)protein are prepared using hybridoma technology. (Kohler et al., Nature256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler etal., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized with G-proteinChemokine Receptor (CCR5) polypeptide or, more preferably, with asecreted G-protein Chemokine Receptor (CCR5) polypeptide-expressingcell. Such polypeptide-expressing cells are cultured in any suitabletissue culture medium, preferably in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the G-protein ChemokineReceptor (CCR5) polypeptide.

Alternatively, additional antibodies capable of binding to G-proteinChemokine Receptor (CCR5) polypeptide can be produced in a two-stepprocedure using anti-idiotypic antibodies. Such a method makes use ofthe fact that antibodies are themselves antigens, and therefore, it ispossible to obtain an antibody which binds to a second antibody. Inaccordance with this method, protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the G-protein Chemokine Receptor (CCR5) protein-specificantibody can be blocked by G-protein Chemokine Receptor. Such antibodiescomprise anti-idiotypic antibodies to the G-protein Chemokine Receptor(CCR5) protein-specific antibody and are used to immunize an animal toinduce formation of further G-protein Chemokine Receptor (CCR5)protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation of Antibody Fragments Directed Against G-Protein ChemokineReceptor (CCRS) from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstG-protein Chemokine Receptor (CCR5) to which the donor may or may nothave been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated hereinby reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Example 16 Production of G-Protein Chemokine Receptor (CCR5) Protein orLigand for High-Throughput Screening Assays

The following protocol produces a supernatant containing a solubleG-protein Chemokine Receptor (CCR5) polypeptide or a G-protein ChemokineReceptor (CCR5) ligand to be tested. Ligands of the G-protein ChemokineReceptor (CCR5) include MIP-1α, MIP-1β, MCP-1, MCP-2, MCP-3, MCP-4,Eotaxin, RANTES, and HIV. This supernatant can then be used in theScreening Assays described in Examples 18-25.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM (Dulbecco's Modified Eagle Medium) (with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS (14-503FBiowhittaker)/1× Penstrep (17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples8-10, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a 12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37° C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl₂ (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃₋₉H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO₄; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; I00 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamineand 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1 L DMEM fora 10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degree C. for 45 or 72 hours depending on the mediaused: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 18-25.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the G-protein Chemokine Receptor (CCR5) polypeptide directly(e.g., as a secreted, soluble, or membrane associated protein) or theG-protein Chemokine Receptor (CCR5) ligand directly, or by the G-proteinChemokine Receptor (CCR5) ligand inducing expression of other proteins,which are then secreted into the supernatant. Thus, the inventionfurther provides a method of identifying the protein in the supernatantcharacterized by an activity in a particular assay.

Example 17 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable 3 below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO: 11)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table 3 below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified.

TABLE 3 JAKs STATS Ligand tyk2 Jak1 Jak2 Jak3 GAS(elements) or ISRE IFNfamily IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) I1-10 + ? ? − 1, 3 gp130 family IL-6(Pleiotrophic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) I1-11(Pleiotrophic) ? + ? ? 1, 3 OnM(Pleiotrophic)? + + ? 1, 3 LIF(Pleiotrophic) ? + + ? 1, 3 CNTF(Pleiotrophic) −/+ + + ?1, 3 G-CSF(Pleiotrophic) ? + ? ? 1, 3 IL-12(Pleiotrophic) + − + + 1, 3g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid)− + − + 6 GAS(IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GASIL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 18-19, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris: 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′ (SEQ ID NO: 12)

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′:GCGGCAAGCTTTTGCAAAGCCTAGGC:3′ (SEQ IDNO: 13)

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2-.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAA ATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCITTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO: 14)

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 18-19.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NFK-B and EGR promoter sequences aredescribed in Examples 20 and 21. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 18

High-Throughput Screening Assay for T-cell Activity.

The following protocol is used to assess T-cell activity by identifyingfactors, and determining whether supernatant containing a polypeptide ofthe invention or a ligand thereof proliferates and/or differentiatesT-cells. T-cell activity is assessed using the GAS/SEAP/Neo constructproduced in Example 17. Thus, factors that increase SEAP activityindicate the ability to activate the Jaks-STATS signal transductionpathway. The T-cell used in this assay is Jurkat T-cells (ATCC AccessionNo. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) andMolt4 cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies) (transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing G-protein Chemokine Receptor (CCR5) polypeptidesor G-protein Chemokine Receptor (CCR5) induced polypeptides as producedby the protocol described in Example 16.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and HIl to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48-72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degree C.until SEAP assays are performed according to Example 18. The platescontaining the remaining treated cells are placed at 4 degree C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

The above protocol may be used in the generation of both transient, aswell as stable transfected cells, which would be apparent to those ofskill in the art.

Example 19 High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of G-proteinChemokine Receptor (CCR5) by determining whether G-protein ChemokineReceptor (CCR5) or G-protein Chemokine Receptor (CCR5) ligandproliferates and/or differentiates myeloid cells. Myeloid cell activityis assessed using the GAS/SEAP/Neo construct produced in Example 17.Thus, factors that increase SEAP activity indicate the ability toactivate the Jaks-STATS signal transduction pathway. The myeloid cellused in this assay is U937, a pre-monocyte cell line, although TF-1,HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 17, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e⁷U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 16. Incubate at 37 degree C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 22.

Example 20 High-Throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesis activated through many different signal transduction pathways. One ofthese genes, EGR1 (early growth response gene 1), is induced in varioustissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells by G-protein Chemokine Receptor(CCR5) or a ligand thereof can be assessed.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells by G-protein Chemokine Receptor (CCR5) or aligand thereof can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers:

5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:15)

5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO: 16)

Using the GAS:SEAP/Neo vector produced in Example 17, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat.No. 08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. No. 12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGRISEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 16. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 12, 37 degree C. for 48 to 72 hr. As a positive control, agrowth factor known to activate PC12 cells through EGR can be used, suchas 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold inductionof SEAP is typically seen in the positive control wells. SEAP assay thesupernatant according to Example 22.

Example 21 High-Throughput Screening Assay for T-Cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a widevariety of agents including the inflammatory cytokines IL-1 and TNF,CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure toLPS or thrombin, and by expression of certain viral gene products. As atranscription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm withI-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylatedand degraded, causing NF-KB to shuttle to the nucleus, therebyactivating transcription of target genes. Target genes activated byNF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KB promoter element are used toscreen the supernatants produced in Example 16. Activators or inhibitorsof NF-KB would be useful in treating, preventing, and/or diagnosingdiseases. For example, inhibitors of NF-KB could be used to treat thosediseases related to the acute or chronic activation of NF-KB, such asrheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR basedstrategy is employed. The upstream primer contains four tandem copies ofthe NF-KB binding site (GGGGACTTCCC) (SEQ ID NO: 17), 18 bp of sequencecomplementary to the 5′ end of the SV40 early promoter sequence, and isflanked with an XhoI site:

5′: GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′ (SEQ ID NO:18)

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 19)

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence:

5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTATTTTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO:20)

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAPcassette is removed from the above NF-KB/SEAP vector using restrictionenzymes SalI and NotI, and inserted into a vector containing neomycinresistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted intopGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 withSalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells arecreated and maintained according to the protocol described in Example18. Similarly, the method for assaying supernatants with these stableJurkat T-cells is also described in Example 18. As a positive control,exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11,with a 5-10 fold activation typically observed.

Example 22 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 18-21, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see the table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 904.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 1557.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48250 12.5 49 255 12.75 50 260 13

Example 23 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 degreesC. water bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by a molecule, such as G-protein Chemokine Receptor (CCR5) or aligand thereof, or a molecule induced by G-protein Chemokine Receptor(CCR5), which has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 24 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase (RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether G-protein ChemokineReceptor (CCR5) or a ligand thereof, or a molecule induced by G-proteinChemokine Receptor (CCR5) is capable of activating tyrosine kinasesignal transduction pathways is of interest. Therefore, the followingprotocol is designed to identify such molecules capable of activatingthe tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 16, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na₃VO4, 2 mM Na₄P207 and a cocktail of proteaseinhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4° C. The plate is then placed in a vacuum transfermanifold and the extract filtered through the 0.45 mm membrane bottomsof each well using house vacuum. Extracts are collected in a 96-wellcatch/assay plate in the bottom of the vacuum manifold and immediatelyplaced on ice. To obtain extracts clarified by centrifugation, thecontent of each well, after detergent solubilization for 5 minutes, isremoved and centrifuged for 15 minutes at 4 degree C. at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate (1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degree C. for 20 min. This allows the streptavadin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phosphotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5u/ml))to each well and incubate at 37 degree C. for one hour. Wash the well asabove.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 25 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 24, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 16 for 5-20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by G-protein Chemokine Receptor(CCR5) or a ligand thereof or a molecule induced by G-protein ChemokineReceptor.

Example 26 Method of Determining Alterations in the G-Protein ChemokineReceptor (CCRS) Gene

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO: 1. Suggested PCRconditions consist of 35 cycles at 95 degree C. for 30 seconds; 60-120seconds at 52-58 degree C.; and 60-120 seconds at 70 degree C., usingbuffer solutions described in Sidransky, D., et al., Science 252:706(1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofG-protein Chemokine Receptor (CCR5) is also determined and genomic PCRproducts analyzed to confirm the results. PCR products harboringsuspected mutations in G-protein Chemokine Receptor (CCR5) is thencloned and sequenced to validate the results of the direct sequencing.

PCR products of G-protein Chemokine Receptor (CCR5) are cloned intoT-tailed vectors as described in Holton, T. A. and Graham, M. W.,Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase(United States Biochemical). Affected individuals are identified bymutations in G-protein Chemokine Receptor (CCR5) not present inunaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to G-protein Chemokine Receptor.Genomic clones isolated according to Example 6 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, Cg. et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to theG-protein Chemokine Receptor (CCR5) genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of G-protein Chemokine Receptor (CCR5)(hybridized by the probe) are identified as insertions, deletions, andtranslocations. These G-protein Chemokine Receptor (CCR5) alterationsare used as a diagnostic marker for an associated disease.

Example 27 Method of Detecting Abnormal Levels of G-Protein ChemokineReceptor (CCR5) in a Biological Sample

G-protein Chemokine Receptor (CCR5) polypeptides can be detected in abiological sample, and if an increased or decreased level of G-proteinChemokine Receptor (CCR5) is detected, this polypeptide is a marker fora particular phenotype. Methods of detection are numerous, and thus, itis understood that one skilled in the art can modify the following assayto fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect G-proteinChemokine Receptor (CCR5) in a sample, preferably a biological sample.Wells of a microtiter plate are coated with specific antibodies toG-protein Chemokine Receptor (CCR5), at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described in Example 15. The wells are blocked sothat non-specific binding of G-protein Chemokine Receptor (CCR5) to thewell is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining G-protein Chemokine Receptor. Preferably, serial dilutions ofthe sample should be used to validate results. The plates are thenwashed three times with deionized or distilled water to remove unboundedG-protein Chemokine Receptor.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot G-protein Chemokine Receptor (CCR5) polypeptide concentrationon the X-axis (log scale) and fluorescence or absorbance of the Y-axis(linear scale). Interpolate the concentration of the G-protein ChemokineReceptor (CCR5) in the sample using the standard curve.

Example 28 Formulation

The invention also provides methods of treatment and/or prevention ofdiseases, disorders, and/or conditions (such as, for example, any one ormore of the diseases, disorders, and/or conditions disclosed herein) byadministration to a subject of an effective amount of a Therapeutic. Bytherapeutic is meant a polynucleotides or polypeptides of the invention(including fragments and variants), agonists or antagonists thereof,and/or antibodies thereto, in combination with a pharmaceuticallyacceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE. 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (U.S.A.) 82:3688-3692 (1985); Hwang etal., Proc. Natl. Acad. Sci. (U.S.A.) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892) TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may beadministered in combination with the Therapeutics of the invention,include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™(didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T),EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FIC; structurallyrelated to lamivudine (3TC) but with 3- to 10-fold greater activity invitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related tolamivudine but retains activity against a substantial proportion oflamivudine-resistant isolates; Biochem Pharma); Adefovir (refusedapproval for anti-UV therapy by FDA; Gilead Sciences); PREVEON®(Adefovir Dipivoxil, the active prodrug of adefovir; its active form isPMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC,with activity against AZT/3TC-resistant virus); GW420867X (GlaxoWellcome); ZIAGENTM (abacavir/159U89; Glaxo Wellcome Inc.); CS-87(3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI ofthe HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a nextgeneration NNRTI with activity against viruses containing the K103Nmutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity thanits predecessor delavirdine and is active against K103N mutants;Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivativesof efavirenz, designed to be active against viruses with the K103Nmutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A(naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; AbbottLaboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonistssuch as RANTES (9-68), AOP-RANTES NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetoragonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibitfusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha asVX-497 (Vertex); and myvopholic acids such as CellCept (mycophenolatemofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-αc2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120,gp120/soluble CD4 complex, Delta JR-FL protein, branched syntheticpeptide derived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(genetically modified CC chemokines targetted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies (forexample, anti-CXCR4 antibodies such as the anti-CXCR4 antibody 12G5,anti-CCR5 antibodies such as the anti-CCR5 antibodies 2D7, 5C7, PA8,PA9, PA 10, PA 11, PA 12, and PA14, anti-CD4 antibodies such as theanti-CD4 antibodies Q4120 and RPA-T4, anti-CCR3 antibodies such as theanti-CCR3 antibody 7B11, anti-gp120 antibodies such as the anti-gp120antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tatantibodies, anti-TNF-α antibodies, and monoclonal antibody 33A); arylhydrocarbon (AH) receptor agonists and antagonists such as TCDD,3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, andα-naphthoflavone (WO 98/30213); and antioxidants such asγ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

Additional agents that may be used with Therapeutics of the presentinvention with or without the agents above includeanti-lymphoproliferative agents such as all-trans-retinoic acid(all-trans-RA), IFN-γ, EPOCH, and Cidofovir; inhibitors of angiogenesissuch as thalidomide; cytostatic chemotherapeutic agents such ashydroxyurea; anti-infective agents such as Rifabutin, Isoniazid, andRifampin; and antidementia agents such as LU 02-584 (CPI-1189; CentuarPharmaceuticals Inc.).

Dosages of these various agents are known in the art, and can be foundin, for example, The Physician's Desk Reference and the scientificliterature.

The virus mutates very rapidly due to the error prone reversetranscriptase (RT), thus developing resistance to multiple therapeuticagents. By targeting multiple points in the viral pathway (RT, protease,viral entry and viral neutralization) using combination therapy, thehigh mutation rate should be effectively countered. Thus, Therapeuticsof the invention may be used with combinations of antiretroviral agents,including two-drug, three-drug, four-drug, five drug, six-drug,seven-drug, eight-drug, nine-drug and greater combinations. Suchcombinations of antiretroviral agents may be referred to in theliterature as active antiretroviral therapy (ART), highly activeantiretroviral therapy (HAART), continuous HAART, intermittent HAART,“mega” HAART (more than 4, 5, 6, 7, or 8, and preferably more than 9agents), intensive high-dose multi-drug therapy, early treatmentintensification (ETI), maximally assisted therapy (MAT),self-administered therapy (SAT), subcutaneous recombinant human IL-2 inHIV-infected patients with low CD4+ counts under active antiretroviraltherapy (SILCAAT), and maintenance therapy. Preferably, Therapeutics ofthe invention are used in combination with highly active antiretroviraltherapy. Therapeutics of the invention may also be used in combinationwith adjunct agents, such as those above and otherwise disclosed hereinand those well known in the art, either alone or together withantiretroviral agents.

When combining Therapeutics of the invention with any of the aboveagents or combinations of agents, the doses are adjusted as necessary.NRTIs generally do not require dose adjustments when combined, butNNRTIs and PIs may affect each other's levels and potency. Guidance forsuch dose adjustments and for initiating, continuing, managing,altering, and maintaining antiretroviral therapy in general are wellknown by practitioners and are readily available in, for example,Guidelines for the Use of Antiretroviral Agents In HIV-Infected Adultsand Adolescents, Panel on Clinical Practices for Treatment of HIVInfection, Dept. Health and Human Services and Henry J. KaiserFoundation, Jan. 28, 2000, and <<http://www.hivatis.org>> and otherscientific literature.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRJMETHOPR™-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, REFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIIETHAMINE™, LEUCOVOR™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, REFAMPIP™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with Pyrimethamine™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORALT™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In other embodiments, Therapeutics of the invention may be administeredin combination with porcine or human insulin or mixtures thereof;insulin analogs; recombinant human insulin such as HUMULIN™ andNOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™(tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™(tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™,DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™(gliciazide), GLUCOPHAGE™ (metformin), PRECOSE™ (acarbose), AMARYL™(glimepiride), and ciglitazone; thiazolidinediones (TZDs) such asrosiglitazone, AVANDIA™ (rosiglitazone maleate) ACTOS™ (piogliatazone),and troglitazone; alpha-glucosidase inhibitors; bovine or porcineglucagon; somatostatins such as SANDOSTATIN™ (octreotide); anddiazoxides such as PROGLYCEM™ (diazoxide). In still other embodiments,Therapeutics of the invention are administered in combination with oneor more of the following: a biguamide antidiabetic agent, a glitazoneantidiabetic agent, and a sulfonylurea antidiabetic agent.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 29 Method of Treating Decreased Levels of G-Protein ChemokineReceptor

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polynucleotides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of G-protein Chemokine Receptor (CCR5) in anindividual can be treated by administering a G-protein ChemokineReceptor (CCR5) agonist, preferably in the secreted form. Thus, theinvention also provides a method of treatment of an individual in needof an increased level of G-protein Chemokine Receptor (CCR5) polypeptidecomprising administering to such an individual a Therapeutic comprisingan amount of G-protein Chemokine Receptor (CCR5) agonist to increase theactivity level of G-protein Chemokine Receptor (CCR5) in such anindividual.

For example, a patient with decreased levels of G-protein ChemokineReceptor (CCR5) polypeptide receives a daily dose 0.1-100 ug/kg of theagonist for six consecutive days. The exact details of the dosingscheme, based on administration and formulation, are provided in Example28.

Example 30 Method of Treating Increased Levels of G-Protein ChemokineReceptor

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production ofG-protein Chemokine Receptor. This technology is one example of a methodof decreasing levels of G-protein Chemokine Receptor (CCR5) polypeptide,preferably a soluble form, due to a variety of etiologies, such ascancer.

For example, a patient diagnosed with abnormally increased levels ofG-protein Chemokine Receptor (CCR5) is administered intravenouslyantisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21days. This treatment is repeated after a 7-day rest period if thetreatment was well tolerated. The formulation of the antisensepolynucleotide is provided in Example 28.

Other methods to decrease G-protein Chemokine Receptor (CCR5) or toinhibit its activity are described herein (such as in Example 57).

Example 31 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing G-protein Chemokine Receptor (CCR5) polypeptides, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask, approximately ten pieces areplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted and the chunks of tissue remain fixed to thebottom of the flask and fresh media (e.g., Ham's F12 media, with 10%FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The DNA encoding G-protein Chemokine Receptor (CCR5) can be amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively as set forth in Example 5. Preferably, the 5′ primercontains an EcoRI site and the 3′ primer includes a HindIII site. Equalquantities of the Moloney murine sarcoma virus linear backbone and theamplified EcoRI and HindIII fragment are added together, in the presenceof T4 DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The ligation mixture isthen used to transform bacteria HB101, which are then plated onto agarcontaining kanamycin for the purpose of confirming that the vectorcontains properly inserted G-protein Chemokine Receptor.

The amphotropic pA317 or GP+aml2 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the G-protein Chemokine Receptor (CCR5) gene is then added tothe media and the packaging cells transduced with the vector. Thepackaging cells now produce infectious viral particles containing theG-protein Chemokine Receptor (CCR5) gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether G-proteinChemokine Receptor (CCR5) protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 32 Gene Therapy Using Endogenous G-Protein Chemokine Receptor(CCR5) Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous G-protein ChemokineReceptor (CCR5) sequence with a promoter via homologous recombination asdescribed, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24,1997; International Publication No. WO 96/29411, published Sep. 26,1996; International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous G-protein Chemokine Receptor (CCR5), flanking thepromoter. The targeting sequence will be sufficiently near the 5′ end ofG-protein Chemokine Receptor (CCR5) so the promoter will be operablylinked to the endogenous sequence upon homologous recombination. Thepromoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousG-protein Chemokine Receptor (CCR5) sequence. This results in theexpression of G-protein Chemokine Receptor (CCR5) in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM +10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the G-protein Chemokine Receptor(CCR5) locus, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digestedwith HindIII. The CMV promoter is amplified by PCR with an XbaI site onthe 5′ end and a BamHI site on the 3′ end. Two G-protein ChemokineReceptor (CCR5) non-coding sequences are amplified via PCR: oneG-protein Chemokine Receptor (CCR5) non-coding sequence (G-proteinChemokine Receptor (CCR5) fragment 1) is amplified with a HindIII siteat the 5′ end and an Xba site at the 3′end; the other G-proteinChemokine Receptor (CCR5) non-coding sequence (G-protein ChemokineReceptor (CCR5) fragment 2) is amplified with a BamHI site at the 5′ endand a HindIII site at the 3′ end. The CMV promoter and G-proteinChemokine Receptor (CCR5) fragments (1 and 2) are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; G-protein ChemokineReceptor (CCR5) fragment 1—XbaI; G-protein Chemokine Receptor (CCR5)fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 33 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) G-protein Chemokine Receptor (CCR5) sequences intoan animal to increase or decrease the expression of the G-proteinChemokine Receptor (CCR5) polypeptide. The G-protein Chemokine Receptor(CCR5) polynucleotide may be operatively linked to a promoter or anyother genetic elements necessary for the expression of the G-proteinChemokine Receptor (CCR5) polypeptide by the target tissue. Such genetherapy and delivery techniques and methods are known in the art, see,for example, W090/11092, WO98/11779; U.S. Pat. Nos. 5,693,622,5,705,151, 5,580,859; Tabata H. et al. (1997) Cardiovasc. Res.35(3):470-479, Chao J et al. (1997) Pharmacol. Res. 35(6):517-522, WolffJ. A. (1997) Neuromuscul. Disord. 7(5):314-318, Schwartz B. et al.(1996) Gene Ther. 3(5):405-411, Tsurumi Y. et al. (1996) Circulation94(12):3281-3290 (incorporated herein by reference).

The G-protein Chemokine Receptor (CCR5) polynucleotide constructs may bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, intestine and the like). TheG-protein Chemokine Receptor (CCR5) polynucleotide constructs can bedelivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the G-protein Chemokine Receptor (CCR5)polynucleotides may also be delivered in liposome formulations (such asthose taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci.772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) whichcan be prepared by methods well known to those skilled in the art.

The G-protein Chemokine Receptor (CCR5) polynucleotide vector constructsused in the gene therapy method are preferably constructs that will notintegrate into the host genome nor will they contain sequences thatallow for replication. Any strong promoter known to those skilled in theart can be used for driving the expression of DNA. Unlike other genetherapies techniques, one major advantage of introducing naked nucleicacid sequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The G-protein Chemokine Receptor (CCR5) polynucleotide construct can bedelivered to the interstitial space of tissues within the an animal,including of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue. Interstitial space of thetissues comprises the intercellular fluid, mucopolysaccharide matrixamong the reticular fibers of organ tissues, elastic fibers in the wallsof vessels or chambers, collagen fibers of fibrous tissues, or that samematrix within connective tissue ensheathing muscle cells or in thelacunae of bone. It is similarly the space occupied by the plasma of thecirculation and the lymph fluid of the lymphatic channels. Delivery tothe interstitial space of muscle tissue is preferred for the reasonsdiscussed below. They may be conveniently delivered by injection intothe tissues comprising these cells. They are preferably delivered to andexpressed in persistent, non-dividing cells which are differentiated,although delivery and expression may be achieved in non-differentiatedor less completely differentiated cells, such as, for example, stemcells of blood or skin fibroblasts. In vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides.

For the naked G-protein Chemokine Receptor (CCR5) polynucleotideinjection, an effective dosage amount of DNA or RNA will be in the rangeof from about 0.05 g/kg body weight to about 50 mg/kg body weight.Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kgand more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,as the artisan of ordinary skill will appreciate, this dosage will varyaccording to the tissue site of injection. The appropriate and effectivedosage of nucleic acid sequence can readily be determined by those ofordinary skill in the art and may depend on the condition being treatedand the route of administration. The preferred route of administrationis by the parenteral route of injection into the interstitial space oftissues. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked G-protein Chemokine Receptor (CCR5) polynucleotideconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The dose response effects of injected G-protein Chemokine Receptor(CCR5) polynucleotide in muscle in vivo is determined as follows.Suitable G-protein Chemokine Receptor (CCR5) template DNA for productionof mRNA coding for G-protein Chemokine Receptor (CCR5) polypeptide isprepared in accordance with a standard recombinant DNA methodology. Thetemplate DNA, which may be either circular or linear, is either used asnaked DNA or complexed with liposomes. The quadriceps muscles of miceare then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The G-protein Chemokine Receptor (CCR5) template DNA isinjected in 0.1 ml of carrier in a 1 cc syringe through a 27 gaugeneedle over one minute, approximately 0.5 cm from the distal insertionsite of the muscle into the knee and about 0.2 cm deep. A suture isplaced over the injection site for future localization, and the skin isclosed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for G-protein Chemokine Receptor (CCR5) protein expression. Atime course for G-protein Chemokine Receptor (CCR5) protein expressionmay be done in a similar fashion except that quadriceps from differentmice are harvested at different times. Persistence of G-proteinChemokine Receptor (CCR5) DNA in muscle following injection may bedetermined by Southern blot analysis after preparing total cellular DNAand HIRT supernatants from injected and control mice. The results of theabove experimentation in mice can be use to extrapolate proper dosagesand other treatment parameters in humans and other animals usingG-protein Chemokine Receptor (CCR5) naked DNA.

Example 34 G-Protein Chemokine Receptor (CCRS) Transgenic Animals

The G-protein Chemokine Receptor (CCR5) polypeptides can also beexpressed in transgenic animals. Animals of any species, including, butnot limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons,monkeys, and chimpanzees may be used to generate transgenic animals. Ina specific embodiment, techniques described herein or otherwise known inthe art, are used to express polypeptides of the invention in humans, aspart of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

In addition to expressing the polypeptide of the present invention in aubiquitous or tissue specific manner in transgenic animals, it wouldalso be routine for one skilled in the art to generate constructs whichregulate expression of the polypeptide by a variety of other means (forexample, developmentally or chemically regulated expression).

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistocherically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of G-protein Chemokine Receptor (CCR5) polypeptides, studyingdiseases, disorders, and/or conditions associated with aberrantG-protein Chemokine Receptor (CCR5) expression, and in screening forcompounds effective in ameliorating such diseases, disorders, and/orconditions.

Example 35 G-Protein Chemokine Receptor (CCR5) Knock-Out Animals

Endogenous G-protein Chemokine Receptor (CCR5) gene expression can alsobe reduced by inactivating or “knocking out” the G-protein ChemokineReceptor (CCR5) gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe G-protein Chemokine Receptor (CCR5) polypeptides. The engineeredcells which express and, in one embodiment, preferably secrete thepolypeptides of the invention can be introduced into the patientsystemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of G-protein Chemokine Receptor (CCR5) polypeptides, studyingdiseases, disorders, and/or conditions associated with aberrantG-protein Chemokine Receptor (CCR5) expression, and in screening forcompounds effective in ameliorating such diseases, disorders, and/orconditions.

Example 36 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay: Purified G-protein Chemokine Receptor (CCR5) protein, ortruncated forms thereof, or purified G-protein Chemokine Receptor (CCR5)ligand is assessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of G-protein Chemokine Receptor (CCR5)protein on purified human tonsillar B cells, measured qualitatively overthe dose range from 0.1 to 10,000 ng/mL, is assessed in a standardB-lymphocyte co-stimulation assay in which purified tonsillar B cellsare cultured in the presence of either formalin-fixed Staphylococcusaureus Cowan I (SAC) or immobilized anti-human IgM antibody as thepriming agent. Second signals such as IL-2 and IL-15 synergize with SACand IgM crosslinking to elicit B cell proliferation as measured bytritiated-thymidine incorporation. Novel synergizing agents can bereadily identified using this assay. The assay involves isolating humantonsillar B cells by magnetic bead (MACS) depletion of CD3-positivecells. The resulting cell population is greater than 95% B cells asassessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 105 B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, loug/mlstreptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150 ul.Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well)with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. Thepositive and negative controls are IL2 and medium respectively.

In Vivo Assay: BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of G-protein Chemokine Receptor (CCR5) protein, ortruncated forms thereof or G-protein Chemokine Receptor (CCR5) ligand.Mice receive this treatment for 4 consecutive days, at which time theyare sacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and G-protein Chemokine Receptor(CCR5) protein-treated spleens identify the results of the activity ofG-protein Chemokine Receptor (CCR5) protein on spleen cells, such as thediffusion of peri-arterial lymphatic sheaths, and/or significantincreases in the nucleated cellularity of the red pulp regions, whichmay indicate the activation of the differentiation and proliferation ofB-cell populations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from G-protein ChemokineReceptor (CCR5) protein-treated mice is used to indicate whetherG-protein Chemokine Receptor (CCR5) protein specifically increases theproportion of ThB+, CD45R(B220)dull B cells over that which is observedin control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andG-protein Chemokine Receptor (CCR5) protein-treated mice.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 37 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times withPBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of G-protein Chemokine Receptor (CCR5) protein(total volume 200 μl). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37° C., plates are spun for 2 min. at1000 rpm and 100 μl of supernatant is removed and stored −20° C. formeasurement of IL-2 (or other cytokines) if effect on proliferation isobserved. Wells are supplemented with 100 μl of medium containing 0.5μCi of ³H-thymidine and cultured at 37° C. for 18-24 hr. Wells areharvested and incorporation of ³H-thymidine used as a measure ofproliferation. Anti-CD3 alone is the positive control for proliferation.IL-2 (100 U/ml) is also used as a control which enhances proliferation.Control antibody which does not induce proliferation of T cells is usedas the negative controls for the effects of G-protein Chemokine Receptor(CCR5) proteins.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 38 Effect of G-Protein Chemokine Receptor (CCR5) on theExpression of MHC Class II, Costimulatory and Adhesion Molecules andCell Differentiation of Monocytes and Monocyte-Derived Human DendriticCells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of G-protein ChemokineReceptor (CCR5) or a ligand thereof or LPS (positive control), washedwith PBS containing 1% BSA and 0.02 mM sodium azide, and then incubatedwith 1:20 dilution of appropriate FITC- or PE-labeled monoclonalantibodies for 30 minutes at 4° C. After an additional wash, the labeledcells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofG-protein Cheniokine Receptor (CCR5) for 24 hours. LPS (100 ng/ml) isadded to the cell culture as positive control. Supernatants from thecell cultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofG-protein Chemokine Receptor (CCR5) or LPS (positive control), washedwith PBS containing 1% BSA and 0.02 mM sodium azide, and then incubatedwith 1:20 dilution of appropriate FITC- or PE-labeled monoclonalantibodies for 30 minutes at 4° C. After an additional wash, the labeledcells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. G-protein Chemokine Receptor (CCR5), agonists, or antagonistsof G-protein Chemokine Receptor (CCR5) can be screened using the threeassays described below. For each of these assays, Peripheral bloodmononuclear cells (PBMC) are purified from single donor leukopacks(American Red Cross, Baltimore, Md.) by centrifugation through aHistopaque gradient (Sigma). Monocytes are isolated from PBMC bycounterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubated atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/milwith increasing concentrations of G-protein Chemokine Receptor (CCR5)and under the same conditions, but in the absence of G-protein ChemokineReceptor. For IL-12 production, the cells are primed overnight with IFN(100 U/ml) in presence of G-protein Chemokine Receptor. LPS (10 ng/ml)is then added. Conditioned media are collected after 24 h and keptfrozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 isthen performed using a commercially available ELISA kit (e.g., R & DSystems (Minneapolis, Minn.)) and applying the standard protocolsprovided with the kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of G-protein Chemokine Receptor(CCR5) are added to the wells in a total volume of 0.2 ml culture medium(RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation,the plates are centrifuged and the medium is removed from the wells. Tothe macrophage monolayers, 0.2 ml per well of phenol red solution (140mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant(200 nM PMA). The plates are incubated at 37° C. for 2 hours and thereaction is stopped by adding 20 μl 1N NaOH per well. The absorbance isread at 610 nm. To calculate the amount of H₂O₂ produced by themacrophages, a standard curve of a H₂O₂ solution of known molarity isperformed for each experiment.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 39 G-Protein Chemokine Receptor (CCR5) Biological Effects

Astrocyte and Neuronal Assays

Recombinant G-protein Chemokine Receptor (CCR5), expressed inEscherichia coli and purified as described above, can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FGF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate G-protein Chemokine Receptor'sactivity on these cells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke, P. et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of G-proteinChemokine Receptor (CCR5) to induce neurite outgrowth can be compared tothe response achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE₂ assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or G-protein Chemokine Receptor (CCR5) with or without IL-1α for24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or G-protein Chemokine Receptor (CCR5) with orwithout IL-1α for 24 hours. The supernatants are collected and assayedfor IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or G-protein ChemokineReceptor (CCR5) for 3 days in basal medium before the addition of AlamarBlue to assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith G-protein Chemokine Receptor.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, G-protein Chemokine Receptor (CCR5) can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofG-protein Chemokine Receptor (CCR5) is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if G-protein Chemokine Receptor (CCR5) acts to prolong thesurvival of dopaminergic neurons, it would suggest that G-proteinChemokine Receptor (CCR5) may be involved in Parkinson's Disease.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 40 The Effect of G-Protein Chemokine Receptor (CCR5) on theGrowth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells EEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. G-proteinChemokine Receptor (CCR5) protein of SEQ ID NO. 2 or SEQ ID NO:22, andpositive controls, such as VEGF and basic FGF (bFGF) are added, atvarying concentrations. On days 4 and 6, the medium is replaced. On day8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that G-proteinChemokine Receptor (CCR5) may proliferate vascular endothelial cells.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 41 Stimulatory Effect of G-Protein Chemokine Receptor (CCR5) onthe Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the calorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or G-protein Chemokine Receptor (CCR5) in0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowedto incubate for 1 hour at 37° C. before measuring the absorbance at 490nm in an ELISA plate reader. Background absorbance from control wells(some media, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 42 Stimulation of Endothelial Migration

This example will be used to explore the possibility that G-proteinChemokine Receptor (CCR5) may stimulate lymphatic endothelial cellmigration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., etal., J. Immunological Methods 1980; 33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 43 Effect of G-Protein Chemokine Receptor (CCR5) on CordFormation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or G-protein Chemokine Receptor (CCR5)(0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr.The numbers and lengths of the capillary-like chords are quantitatedthrough use of the Boeckeler VIA-170 video image analyzer. All assaysare done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-estradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 44 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of G-protein Chemokine Receptor (CCR5) ora ligand thereof to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese quail (Cotumix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old quailembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, IL) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 45 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of G-protein Chemokine Receptor (CCR5)measures the ability of an existing capillary network to form newvessels in an implanted capsule of murine extracellular matrix material(Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C.and the mixture is then injected subcutaneously in mice where itsolidifies. After 7 days, the solid “plug” of Matrigel is removed andexamined for the presence of new blood vessels. Matrigel is purchasedfrom Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with G-protein Chemokine Receptor (CCR5) at 150 ng/mlat 4 degree C. and drawn into cold 3 ml syringes. Female C57Bl/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffin andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 46 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of G-protein Chemokine Receptor (CCR5) onischemia, a rabbit hindlimb ischemia model is created by surgicalremoval of one femoral arteries as described previously (Takeshita, S.et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoralartery results in retrograde propagation of thrombus and occlusion ofthe external iliac artery. Consequently, blood flow to the ischemic limbis dependent upon collateral vessels originating from the internal iliacartery (Takeshita, S. et al. Am J. Pathol 147:1649-1660 (1995)). Aninterval of 10 days is allowed for post-operative recovery of rabbitsand development of endogenous collateral vessels. At 10 daypost-operatively (day 0), after performing a baseline angiogram, theinternal iliac artery of the ischemic limb is transfected with 500 mgnaked G-protein Chemokine Receptor (CCR5) expression plasmid by arterialgene transfer technology using a hydrogel-coated balloon catheter asdescribed (Riessen, R. et al. Hum Gene Ther. 4:749-758 (1993); Leclerc,G. et al. J. Clin. Invest. 90: 936-944 (1992)). When G-protein ChemokineReceptor (CCR5) is used in the treatment, a single bolus of 500 mgG-protein Chemokine Receptor (CCR5) protein or control is delivered intothe internal iliac artery of the ischemic limb over a period of 1 min.through an infusion catheter. On day 30, various parameters are measuredin these rabbits: (a) BP ratio—The blood pressure ratio of systolicpressure of the ischemic limb to that of normal limb; (b) Blood Flow andFlow Reserve—Resting FL: the blood flow during undilated condition andMax FL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 47 Peripheral Arterial Disease Model

Angiogenic therapy using G-protein Chemokine Receptor (CCR5) is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) G-protein Chemokine Receptor (CCR5) protein, in a dosage range of 20mg—500 mg, is delivered intravenously and/or intramuscularly 3 times(perhaps more) per week for 2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of G-protein ChemokineReceptor (CCR5) expression and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 48 Ischemic Myocardial Disease Model

G-protein Chemokine Receptor (CCR5) is evaluated as a potent mitogencapable of stimulating the development of collateral vessels, andrestructuring new vessels after coronary artery occlusion. Alteration ofG-protein Chemokine Receptor (CCR5) expression is investigated in situ.The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) G-protein Chemokine Receptor (CCR5) protein, in a dosage range of 20mg—500 mg, is delivered intravenously and/or intramuscularly 3 times(perhaps more) per week for 2-4 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 49 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby G-Protein Chemokine Receptor

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of G-protein Chemokine Receptor (CCR5) to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca, Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μlof diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca, Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5). 5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example test activity in G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 50 Methods of Inhibiting G-protein Coupled Receptor ActivityUsing Transmembrane Fragments

WO 94/05695 and U.S. Pat. No. 5,508,384 set forth sequences oftransmembrane regions for 74 GPCRs. The WO 94/05695 patent publicationdescribes and claims polypeptides corresponding to fragments orhomologous sequences of GPCRs which can bind a GPCR ligand or which canmodulate ligand binding. Both references disclose that a membranespanning fragment of the third TM domain of the dopamine D2 receptorspecifically bound a ligand of the intact receptor in a simple, smallunilamellar vesicle model. The fragment used was terminated with alysine (which is positively charged at physiological pH) at one end andwith an aspartic acid (which is negatively charged at physiological pH)at the other. This peptide would not be expected to insert readily intoa biological membrane.

In contrast, this example relates to modulating, especially inhibiting,biological activities of G-protein Chemokine Receptor (CCR5) by exposingit to molecules which interfere with correct receptor assembly. Inparticular, synthetic, isolated and/or recombinant peptides, fragmentsand/or consensus peptides of the transmembrane domain of G-proteinChemokine Receptor (CCR5) inhibit G-protein Chemokine Receptor (CCR5)mediated signal transduction. Charged residues may be added at oneterminus to promote correct orientation of the peptide in the membrane.In particular, addition of two negatively charged residues, such as Asp,at the extracellular terminus of the fragment enhances antagonistactivity.

Fragments of the transmembrane domain can be synthesized by flow-throughsolid phase peptide synthesis on 432A Applied Biosystems PeptideSynthesizer utilizing Fmoc amino acid derivatives. To overcomeaggregation that may occur during synthesis of the peptides and that maylead to blockage of the growing peptide chain, FmocHmb derivatives ofAla, Val, and Leu are introduced. Charged residues are added to thepeptide termini to assure a proper orientation of the peptides duringpenetration into the cellular membrane, and to improve solubility ofhydrophobic peptides. Purity of the peptides is assessed by reversephase HPLC and the structures are confirmed by matrix-assistedlaser-desorption time-of-flight (MALDI-TOF) mass spectrometry (Tarasovaet al., Ad. Exp. Med. Biol., Plenum Press, NY, pp. 201-206 (1998).)

The antagonistic effect of the fragments is tested on human kidneycarcinoma (HEK) cells stably expressing the G-protein ChemokineReceptor. RANTES is used as the agonist. Cells grown on Nunc cover glasschamber slides are incubated with 1 μM Fura-2/AM for 20 min. in a CO₂incubator, rinsed with PBS, and mounted on the stage of a Zeiss Axiovertinverted microscope. [Ca²⁺]i measurements are performed using anAttofluor digital imaging system (Atto Instruments). Fluorescence ismonitored by an intensified CCD camera using a 505 cut-off filter.Calibrations of [Ca²⁺]i is performed using Ca²⁺ standards containing 1μM Fura. The antagonist activity of the fragments is further optimizedas described in Examples 1-4 of WO 99/43711.

The antagonist activity of the fragments is also tested by the abilityto inhibit G-protein Chemokine Receptor-HIV cell fusion, and the abilityto inhibit binding of a labeled ligand of G-protein Chemokine Receptor(CCR5), by methods well-known in the art and as described for CXCR4 inWO 99/43711.

Example 51 Herpes Virus Immortalized T Cells which Express the G-ProteinChemokine Receptor (CCRS)

The construction of a Herpes Virus immortalized T cell line whichexpresses the G-protein Chemokine Receptor (CCR5) is described in Vella,et al., J. Virol. Methods 79:51-63 (1999). This or a similar cell lineis useful to assay agonists and antagonists in the methods disclosedherein.

Example 52 Isolation of CCR5 Ligands and Anti-CCR5Antibodies

A general method for solubilizing CCR5 in its native state that may beused in ligand and antibody screening assays is disclosed in Mirzabekovet al., J. Biol. Chem. 274:28745-50 (1999). A method of selecting CCR5antibody from a phage display library of human antibodies is disclosedin Osbourn et al., Nat. Biotechnol. 16:778-81 (1998). Lee et al.disclose that the epitope recognized by the CCR5-specific antibody 2D7is a preferred target for antibodies to inhibit HIV entry. Lee et al. J.Biol. Chem. 274:9617-26 (1999). Other methods of screening for ligandsand antibodies are well known in the art and are described herein.

Example 53 Assays for Antibody Neutralization

A cell-line based assay for measuring neutralization of H1V-1 byantibodies is disclosed in Trkola et al., J. Virol. 73:8966-74 (1999).An assay for HIV-neutralizing antibody and screen for a molecule thatinhibits HV binding or entry at any stage is disclosed in Boritz et al.,J. Virol. 73:6937-45 (1999). A method for analyzing co-receptorinhibition is disclosed in Klasse et al., J. Virol. 73:7453-66 (1999).Additional methods of assaying neutralization of HIV entry, fusion,replication, etc., are well known in the art and disclosed herein.

Example 54 Generation of Anti-G-Protein Chemokine Receptor (CCR5)Antibodies Using Xenomouse™ Strains

Xenomouse™ strains of mice engineered to express a repertoire of humanIgM/Kappa or IgG2/kappa antibodies were obtained from Abgenix, Inc,(Fremont, Calif.). Groups of mice were immunized according to thefollowing schedules:

Immunization Schedule 1 (XF3 Fusion):

Xenomouse™ mice (n=5) were initially injected in the base of the tailwith 100 micrograms in PBS of a DNA plasmid expression vector encodingthe full-length G-protein Chemokine Receptor (CCR5) gene (CCR5 pcDNA3T).This was followed by three sub-cutaneous injections given at two weekintervals, each consisting of 10 million CHO cells transfected with aCCR5 expression vector (hereinafter “CCR5CHO cells”) in incompleteFreund's adjuvant. The animals were allowed to rest for 12 weeks andthen given two more sub-cutaneous injections separated by two weeks,each consisting of 10 million NSO cells transfected with a CCR5expression vector (hereinafter “CCR5NSO cells”) in incomplete Freund'sadjuvant. Three days after the last injection. Mice were sacrificed andspleen and/or lymph node cells were collected for the purposes ofgenerating hybridomas. Hybridomas generated from this fusion arereferred to as “XF3.----” (Table 4).

Immunization Schedule 2 (XF6 Fusion):

Xenomouse™ mice (n=5) were initially injected intraperitoneally with 7million CCR5CHO cells in complete Freund's adjuvant. This was followedby six intraperitoneal injections given at two week intervals, eachconsisting of 10 million CCR5CHO cells. The animals were allowed to restfor 5 weeks and then given two more intraperitoneal injections separatedby two weeks, each consisting of 10 million CCR5NSO cells in incompleteFreund's adjuvant. Three days after the last injection. Mice weresacrificed and spleen and/or lymph node cells were collected for thepurposes of generating hybridomas. Hybridomas generated from this fusionare referred to as “XF6.----” (Table 4).

Immunization Schedule 3 (XF7 Fusion):

Xenomouse™ mice (n=5) were initially injected in the base of the tailwith 7 million CCR5CHO cells in complete Freund's adjuvant. This wasfollowed by six additional injections in the base of the tail given attwo week intervals, each consisting of 10 million CCR5CHO cells. Theanimals were allowed to rest for 5 weeks and then given two moreinjections in the base of the tail separated by two weeks, eachconsisting of 10 million CCR5NSO cells in incomplete Freund's adjuvant.Three days after the last injection, mice were sacrificed and spleenand/or lymph node cells were collected for the purposes of generatinghybridomas. Hybridomas generated from this fusion are referred to as“XF7.----” (Table 4).

Immunization Schedule 4 (XF11 Fusion):

Xenomouse™ mice (n=5) were immunized via injection in the footpads givenat two week intervals. Each immunization consisted of a total of 10million CCR5NSO cells in RIBI adjuvant. A total of eight suchimmunization were administered. Three days after the last injection,mice were sacrificed and spleen and/or lymph node cells were collectedfor the purposes of generating hybridomas. Hybridomas generated fromthis fusion are referred to as “XF11.----” (Table 4).

Immunization Schedule 5 (XF12 Fusion):

Xenomouse™ mice (n=5) were initially immunized with 10 million CCR5. NSOcells in complete Freund's adjuvant administered via a combination ofintraperitoneal and subcutaneous routes. This was followed by sixadditional immunizations, given at two week intervals, each consistingof 10 million CCR5CHO cells in incomplete Freund's adjuvant, alsoadministered via a combination of intraperitoneal and subcutaneousroutes. One animal was sacrificed for fusion three days after the thirdbooster immunization in incomplete Freund's adjuvant. Three days afterthe last injection, the remaining mice were sacrificed and spleen and/orlymph node cells were collected for the purposes of generatinghybridomas. Hybridomas generated from this fusion are referred to as“XF12.----” (Table 4).

Immunization Schedule 6 (XF27/28 Fusion):

CCR5/XF27 & 28 FP Immunization Schedule Animals 20 mice Day No. ActionAntigen Amount Volume Adjuvant Day 1 Inject CCR5 NSO cells FP 5 × 10⁶cells/ms 50 μl 1:1 PBS/RIBI Day 14 Inject CCR5 NSO cells FP 5 × 10⁶cells/ms 50 μl 1:1 PBS/RIBI Day 28 Inject CCR5 NSO cells FP 5 × 10⁶cells/ms 50 μl 1:1 PBS/RIBI Day 38 Inject CCR5 NSO cells FP 5 × 10⁶cells/ms 50 μl PBS Day 41 Fusion

Hybridomas were generated according to protocols which are commonlyknown in the art. The fusion partner used to generate these hybridomaswas P3×63-AG8.653 purchased from the ATCC, Batch F11545.

Antibodies produced by these hybridomas were screened for the ability tobind CCR5 by both ELISA and FACS screening.

Membrane ELISA Screening for Anti-G-Protein Chemokine Receptor (CCR5)Specific Antibodies

Plasma Membrane Preparation. Plasma membranes from CCR5 CHO cells andvector control transfected CHO cells were prepared. Briefly between 10⁸to 10⁹ CCR5CHO or CHO cells were suspended in 40-50 milliliters of cold12 mM Tris, pH 7.5, 250 mM sucrose. Cells were lysed on ice, byhomogenization using a variable speed electric homogenator. Cell lysiswas confirmed by microscopy. The cell homogenate was centrifuged at270×g, for 10 minutes at 4° C. The supernatant (containing the plasmamembranes) was collected while the pellet (containing the nuclearfraction was discarded. Next, the supernatant was centrifuge at 8000×g,for 10 minutes at 4° C. Again, the supernatant (containing the plasmamembranes) was collected while the pellet (containing the mitochondrialand lysosomal fractions) was discarded. The plasma membranes were thenspun out of the supernatant by centrifugation in an ultracentrifuge at100,000×g, for 60 minutes at 4° C. The supernatant was discarded. Thepelleted plasma membranes were resuspended in approximately 1 ml of PBS.After resuspension the volume of plasma membranes was brought up to 5-10ml with additional PBS. The membrane solution was kept on ice andsonicated (on ice) until a uniform solution was obtained. Care was takennot to overheat the solution during sonication. The plasma membraneprotein concentration was determined using the BCA protein determinationkit available from Pierce Chemical Company (Rockford Ill.). The plasmamembranes were stored −70° C. until use.

Membrane ELISA. Immulon 4 plates (Dynex) were coated with 50 microlitersof either CCR5CHO or vector control transfected CHO plasma membranes(membrane solutions were at a concentration of 20 microgram/milliliter)overnight at 4° C. The next morning, the plates were washed three timeswith PBST (PBST=PBS containing 0.01% Tween20). The plates were thenblocked for 1 hr at room temperature with 200 microliters/well of 3%BSA/PBS. The blocking solution was removed from the plates and 50microliters/well of samples (hybridoma supernatant) and controls wereadded to the plates and incubated for 2 hours at room temperature orovernight at 4° C. Each sample/control was tested for binding to bothCCR5CHO membranes as well as vector control transfected CHO membranes.Next the plates were washed three times with PBST. Following washing, 50microliters/well of secondary antibody (Vector Goat anti-Human IgG (H+L)at 0.25 ug/ml in 0.1% BSA/PBST+1% Goat Serum) was added to the wells andincubated for 1 hour at room temperature. While the plates wereincubating, the ABC reagent (Vector Laboratories) was prepared. Theplate was then three times with PBST. Next, 50 microliters/well ofdiluted ABC were added to the plates and incubated for 30 minutes atroom temperature. The plates were then washed 6 times PBST. 100microliters/well of TMB reagent (Sigma Chemical Company, St. Louis, Mo.)were added to the wells and incubated for 10 minutes at roomtemperature. The reaction was then stopped by adding 25 microliters/wellof 2M H₂SO₄. The plate was read at 405 nm.

Results. 238 hybridomas showed binding to membranes of CCR5 CHO cells,of which approximately one half showed increased binding to CCR5CHOmembranes compared to vector control transfected CHO membranes. 217(Table 4) of these hybridomas were expanded and the membrane ELISA wasrepeated. Results from the second screening demonstrated that theresults of this screening procedure were reproducible.

TABLE 4 Hybridomas that secrete antibodies that bind CCR5 CHO membranesXF3 Fusion XF6 Fusion XF7 Fusion XF11 Fusion XF12 Fusion XF3.10B8XF6.LNG9 XF7.1A10 XF11.1E2 XF12.10B2 XF3.10C4 XF6.4D11 XF7.2E10 XF11.1E6XF12.11F5 XF3.10G4 XF6.LNC6 XF7.2A2 XF11.1A11 XF12.12B11 XF3.10H12XF6.LNF6 XF7.2C4 XF11.1A2 XF12.13H6 XF3.11B5 XF6.LNC11 XF7.3A5 XF11.1A8XF12.15B11 XF3.13D3 XF6.LND9 XF7.3H1 XF11.1B10 XF12.1B8 XF3.14E12XF6.LNF2 XF7.3H2 XF11.1B12 XF12.2E1 XF3.15C2 XF7.3H8 XF11.1B4 XF12.2E12XF3.15F6 XF7.4E8 XF11.1B7 XF12.2H5 XF3.2A3 XF7.4E9 XF11.1B9 XF12.2H8XF3.2E5 XF7.4A6 XF11.1C1 XF12.3E2 XF3.3H1 XF7.4B2 XF11.1C7 XF12.3A9XF3.4B6 XF7.4G3 XF11.1D10 XF12.3C2 XF3.4C5 XF7.4G7 XF11.1D8 XFL2.3G11XF3.5F1 XF7.4H4 XF11.1F8 XF12.4A8 XF3.6A1 XF7.4H7 XF11.1G11 XF12.4G7XF3.6A2 XF7.5A1 XF11.1G8 XF12.5B10 XF3.6H11 XF7.5B8 XF11.1H7 XF12.5B11XE3.7C11 XF7.5B9 XF11.2E4 XF12.5F1 XF3.8D5 XF7.5H8 XF11.2E5 XF12.5H1XF3.8G10 XF7.6B11 XF11.2B9 XF12.6B12 XF3.9G3 XF7.6B12 XF11.2C9 XF12.6H1XF3.LNA2 XF7.6B3 XF11.2D1 XF12.6H7 XF3.LNB12 XF7.6D12 XF11.2D10XF12.7F12 XF3.LNC10 XF7.6D3 XF11.2D11 XF12.LND11 XF3.LNC11 XF7.6D7XF11.2D5 XF3.LNC2 XF7.7A9 XF11.2D8 XF3.LNC3 XF7.7B6 XF11.2F3 XF3.LNC4XF7.7C11 XF11.2F5 XF3.LNC6 XF7.7C4 XF11.2F6 XF3.LND9 XF7.7E8 XF11.2F7XF3.LNE7 XF7.7F8 XF11.2F8 XF3.LNF1 XF7.7G4 XF11.2F9 XF3.LNH5 XF7.LN1B1XF11.2G11 XF7.LN1B7 XF11.2G4 XF7.LN1D10 XF11.2G6 XF7.LN1D11 XF11.2G8XF7.LN1D9 XF11.2G9 XF7.LN1E10 XF11.2H10 XF7.LN1E11 XF11.2H2 XF7.LN1E12XF11.2H4 XF7.LN2A11 XF11.2H8 XF7.LN2A7 XF11.3A3 XF11.3B10 XF11.3B9XF11.3C3 XF11.3C6 XF11.3C7 XF11.3D1 XF11.3D12 XF11.3D2 XF11.3D3 XF11.3F4XF11.3G12 XF11.3G2 XF11.3G3 XF11.3H7 XF11.4E11 XF11.4A1 XF11.4A5XF11.4B10 XF11.4B12 XF11.4B3 XF11.4B4 XF11.4C10 XF11.4C12 XF11.4C4XF11.4D10 XF11.4D12 XF11.4D3 XF11.4D4 XF11.4D5 XF11.4F11 XF11.5E2XF11.5A2 XF11.5C10 XF11.5D12 XF11.5F2 XF11.5F3 XF11.5G10 XF11.5G11XF11.5G4 XF11.5G5 XF11.5G6 XF11.5H1 XF11.5H4 XF11.5H15 XF11.6E12XF11.6E4 XF11.6E7 XF11.6A3 XF11.6A5 XF11.6B3 XF11.6B4 XF11.6B9 XF11.6C11XF11.6C5 XF11.6D7 XF11.6D8 XF11.6D9 XF11.6F2 XF11.6F9 XF11.6G1 XF11.6G6XF11.6H11 XF11.6H2 XF11.6H4 XF11.6H7FACS Screening for Anti-G-Protein Chemokine Receptor (CCR5) SpecificAntibodies

G-Protein Chemokine Receptor (CCR5) transfected or vector controltransfected CHO cells were harvested, washed with FACS buffer (PBS with0.1% NaN3 and 0.1% BSA). One million cells in 100 ul were dispensed toFACS tubes (Falcon 2052). 10 microliters of hybridoma supernatant wasadded to each tube and incubated for 20 min at 4 C. Each supernatant wasanalyzed for binding to both CCR5CHO and vector control transfected CHOcells. Cells were washed, and resuspended in 100 microliters of FACSbuffer and 10 microliters of biotinylated Goat anti-Human IgG (H+ L)(Vector) at 1 microgram/milliliter was added to the tubes and incubated20 min at 4 C. Cells were washed, resuspended in 100 microliters of FACSbuffer and 5 microliters of Streptavidin PE (DAKO) was added followed bya 10 minute incubation at 4 degrees C. Cells were washed, resuspended in200 microliters of FACS buffer containing 0.5 micrograms/milliliter ofpropidium iodide and analyzed on FACScan (Becton Dickinson).

Results. Of the 217 hybridomas supernatants screened by FACS analysis,XF11.1D8, XF11.4D10, XF11.4C4, XF11.5H1, and XF11.1G8 were identified asshowing significantly increased binding to CCR5CHO compared to vectorcontrol transfected CHO cells.

Additional hybridomas were produced and their supernatents were screenedfor anti-G-protein Chemokine Receptor (CCR5) specific antibodies. Anumber of these were also found to have significantly increased bindingto CCR5 compared to controls. They are described herein as preferredantibodies, and are listed in Table 2.

Example 55 Identification and Cloning of VH and VL Domains

One method to identify and clone VH and VL domains from cell linesexpressing a particular antibody is to perform PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may lysed in the TRIzol® reagent(Life Technologies, Rockville. MD) and extracted with one fifth volumeof chloroform. After addition of chloroform, the solution is allowed toincubate at room temperature for 10 minutes, and the centrifuged at14,000 rpm for 15 minutes at 4° C. in a tabletop centrifuge. Thesupernatant is collected and RNA is precipitated using an equal volumeof isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000rpm for 15 minutes at 4° C. in a tabletop centrifuge. Followingcentrifugation, the supernatant is discarded and washed with 75%ethanol. Following washing, the RNA is centrifuged again at 800 rpm for5 minutes at 4° C. The supernatant is discarded and the pellet allowedto air dry. RNA is the dissolved in DEPC water and heated to 60° C. for10 minutes. Quantities of RNA can determined using optical densitymeasurements.

cDNA may be synthesized, according to methods well-known in the art,from 1.5-2.5 micrograms of RNA using reverse transciptase and randomhexamer primers. cDNA is then used as a template for PCR amplificationof VH and VL domains. Primers used to amplify VH and VL genes are shownin Table 5. Typically a PCR reaction makes use of a single 5′ primer anda single 3′ primer. Sometimes, when the amount of available RNA templateis limiting, or for greater efficiency, groups of 5′ and/or 3′ primersmay be used. For example, sometimes all five VH-5′ primers and all JH3′primers are used in a single PCR reaction. The PCR reaction is carriedout in a 50 microliter volume containing 1×PCR buffer, 2 mM of eachdNTP, 0.7 units of High Fidelity Taq polymerase, 5′ primer mix, 3′primer mix and 7.5 microliters of cDNA. The 5′ and 3′ primer mix of bothVH and VL can be made by pooling together 22 pmole and 28 pmole,respectively, of each of the individual primers. PCR conditions are: 96°C. for 5 minutes; followed by 25 cycles of 94° C. for 1 minute, 50° C.for 1 minute, and 72° C. for 1 minute; followed by an extension cycle of72° C. for 10 minutes. After the reaction is completed, sample tubeswere stored 4° C.

TABLE 5 Primer Sequences Used to Amplify VH and VL domains. Primer nameSEQ ID NO Primer Sequence (5′-3′) VH Primers Hu VH1-5′ 23CAGGTGCAGCTGGTGCAGTCTGG Hu VR2-5′ 24 CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5′25 GAGGTGCAGCTGGTGGAGTCTGG Hu VH4-5′ 26 CAGGTGCAGCTGCAGGAGTCGGG HuVH5-5′ 27 GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5′ 28 CAGGTACAGCTGCAGCAGTCAGGHu JH1,2-5′ 29 TGAGGAGACGGTGACCAGGGTGCC Hu JH3-5′ 30TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5′ 31 TGAGGAGACGGTGACCAGGGTTCC HuJH6-5′ 32 TGAGGAGACGGTGACCGTGGTCCC VL Primers Hu Vkappa1-5′ 33GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5′ 34 GATGTTGTGATGACTCAGTCTCC HuVkappa2b-5′ 35 GATATTGTGATGACTCAGTCTCC Hu Vkappa3-5′ 36GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5′ 37 GACATCGTGATGACCCAGTCTCC HuVkappa5-5′ 38 GAAACGACACTCACGCAGTCTCC Hu Vkappa6-5′ 39GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5′ 40 CAGTCTGTGTTGACGCAGCCGCC HuVlambda2-5′ 41 CAGTCTGCCCTGACTCAGCCTGC Hu Vlambda3-5′ 42TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5′ 43 TCTTCTGAGCTGACTCAGGACCC HuVlambda4-5′ 44 CACGTTATACTGACTCAACCGCC Hu Vlambda5-5′ 45CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5′ 46 AATTTTATGCTGACTCAGCCCCA HuJkappa1-3′ 47 ACGTTTGATTTCCACCTTGGTCCC Hu Jkappa2-3′ 48ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-3′ 49 ACGTTTGATATCCACTTTGGTCCC HuJkappa4-3′ 50 ACGTTTGATCTCCACCTTGGTCCC Hu Jkappa5-3′ 51ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-3′ 52 CAGTCTGTGTTGACGCAGCCGCC HuJlambda2-3′ 53 CAGTCTGCCCTGACTCAGCCTGC Hu Jlambda3-3′ 54TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-3′ 55 TCTTCTGAGCTGACTCAGGACCC HuJlambda4-3′ 56 CACGTTATACTGACTCAACCGCC Hu Jlambda5-3′ 57CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-3′ 58 AATTTTATGCTGACTCAGCCCCA

TABLE 6 Anti-CCR5 Antibodies XF11.1D8, XF22.3C9 (e.g., XF22.3C9.6), andXF22.9E6 VH VH VL VL Hybridoma DNA protein AAs of AAs of AAs of DNAprotein AAs of AAs of AAs of ATCC Cell Line/ SEQ SEQ ID VH VH VH SEQ IDSEQ ID VL VL VL Deposit ATCC Deposit Antibody ID NO: NO: CDR1 CDR2 CDR3NO: NO: CDR1 CDR2 CDR3 Number Date XF11.1D8 59 60 31-35 50-65  98-110 6162 24-35 51-57 90-98 PTA-3030 Feb. 7, 2001 XF22.3C9 63 64 31-35 50-68102-115 65 66 24-34 50-56 89-97 PTA-3702 Sep. 12, 2001 XF22.9E6 67 6831-35 50-65  98-115 69 70 24-35 51-57 90-98 PTA-3859 Nov. 14, 2001

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands ofthe expected sizes (˜506 base pairs for VH domains, and 344 base pairsfor VL domains) can be cut out of the gel and purified using methodswell known in the art. Purified PCR products can be ligated into a PCRcloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.).Individual cloned PCR products can be isolated after transfection of E.coli and blue/white color selection. Cloned PCR products may then besequenced using methods commonly known in the art. The polynucleotideand amino acid sequences of the VH and VL domains of anti-CCR5antibodies XF11.1D8, XF22.3C9.6, and XF22.9E6 are shown in FIGS. 4, 5,and 6 (see also, Table 6).

The PCR bands containing the VH domain and the VL domains can also beused to create full-length Ig expression vectors. VH and VL domains canbe cloned into vectors containing the nucleotide sequences of a heavy(e.g., human IgG1 or human IgG4) or light chain (human kappa or humanlambda) constant regions such that a complete heavy or light chainmolecule could be expressed from these vectors when transfected into anappropriate host cell. Further, when cloned heavy and light chains areboth expressed in one cell line (from either one or two vectors), theycan assemble into a complete functional antibody molecule that issecreted into the cell culture medium. Methods using polynucleotidesencoding VH and VL antibody domain to generate expression vectors thatencode complete antibody molecules are well known within the art.

Example 56 Immunofluorescence Assay

The following immunofluorescence protocol may be used, for example, toverify G-protein Chemokine Receptor (CCR5) expression on cells, or tocheck for the presence of one or more antibodies that bind G-proteinChemokine Receptor (CCR5) expressed on the surface of cells. Briefly,Lab-Tek II chamber slides are coated at 4° C. overnight with 10micrograms/milliliter of bovine collagen Type II in DPBS containingcalcium and magnesium (DPBS++). The slides are then washed twice withcold DPBS++ and seeded with 8000 CHO—CCR5 or CHO pC4 transfected cellsin a total volume of 125 microliters and incubated at 37° C. in thepresence of 95% oxygen/5% carbon dioxide. The culture medium is gentlyaspirated out and the adhering cells are washed twice with DPBS++ atambient temperature. The slides are blocked with DPBS++ containing 0.2%BSA (blocker) at 0-4° C. for one hour. The blocking solution is gentlyaspirated out and 125 microliters of antibody containing solution (anantibody containing solution may be, for example, a hybridoma culturesupernatant which is usually used undiluted, or serum/plasma which isusually diluted—around a 1/100 dilution). The slides are incubated for 1hour at 0-4° C. Antibody solutions are then gently aspirated off and thecells are washed 5 times with 400 microliters of ice cold blockingsolution. Next. 125 microliters of a 1 microgram/milliliter of rhodaminelabeled secondary antibody (e.g., anti-human IgG) in the blocker isadded to the cells. Again, cells are incubated for 1 hour at 04° C. Thesecondary antibody solution is then aspirated off gently and the cellswashed 3 times with 400 microliters of ice cold blocking solution and 5times with cold DPBS++. The cells are then fixed with 125 microliters of3.7% formaldehyde in DPBS++ for 15 minutes at ambient temperature. Thecells are washed 5 times with 400 μl of DPBS++ at ambient temperature.Finally, the cells are mounted in 50% aqueous glycerol and viewed in afluorescence microscope using rhodamine filters.

Example 57 Western Blotting to Detect Binding to G-Protein ChemokineReceptor (CCR5)

G-protein Chemokine Receptor (CCR5) is a membrane embedded protein. Inorder to perform a western blot on G-protein Chemokine Receptor (CCR5)proteins, cell membranes must first be solubilized. The followingprotocol was worked out by Mirzabekov et al., J. Biol. Chem. 274:28745(1999) which is hereby incorporated in its entirety by reference herein.A single cell suspension of G-protein Chemokine Receptor (CCR5)—CHOcells or pC4-CHO (vector transfected control CHO) cells, is pelleted andresuspended in solubilization buffer composed of 100 mM (NH₄)₂SO₄, 20 mMTris-HCl (pH 7.5) and 1% (w/v) Cymal™-5 (Anatrace Inc., Maumee, Ohio),and protease inhibitor mixture (one tablet of Complete™ (Roche MolecularBiochemicals) per 25 ml. After a 30 minute incubation at 4 C on arocking platform, the samples are centrifuged for 30 minutes at 14,000×gto remove cell debris. G-protein Chemokine Receptor (CCR5) isimmunoprecipitated from the solubilized membrane using, for example, themonoclonal anti-G-protein Chemokine Receptor (CCR5) antibody 2D7described in Wu et al., J. Exp. Med. 186:1373 (1997) conjugated tosepharose beads. Following immunoprecipitation, the beads are washedextensively with solubilization buffer and resuspended in 2×SDS-samplebuffer. Samples are incubated in SDS-sample buffer for 1 hour at 55 Cprior to electrophoresis through an 11% SDS-polyacrylamide gel. Westernblotting on the G-protein Chemokine Receptor (CCR5) samples can then becarried out according to standard protocols known in the art.

Example 58 Western Blotting, Immunoprecipitation, and Purification ofG-protein Chemokine Receptor (CCR5)

The membrane solubilization protocol or Mirzabekov et al described inExample 57 above may also be used to prepare G-protein ChemokineReceptor (CCR5) containing samples for western blotting,immunoprecipitation or purification.

Example 59 BIAcore Analysis of the Affinity of G-protein ChemokineReceptor (CCR5) Binding Polypeptides

Binding of anti-G-protein Chemokine Receptor (CCR5) antibodies toG-protein Chemokine Receptor (CCR5), for example, can be analyzed byBIAcore analysis. Either G-protein Chemokine Receptor (CCR5) (or otherantigen to which one wants to know the affinity of a anti-G-proteinChemokine Receptor (CCR5) antibody) or anti-G-protein Chemokine Receptor(CCR5) antibody can be covalently immobilized to a BLAcore sensor chip(CM5 chip) via amine groups usingN-ethyl-N′-(dimethylaminopropyl)carboiimide/N-hydroxysuccinimidechemistry. Various dilutions of anti-G-protein Chemokine Receptor (CCR5)antibodies or G-protein Chemokine Receptor (CCR5) (or other antigen towhich one wants to know the affinity of a anti-G-protein ChemokineReceptor (CCR5) antibody), respectively are flowed over the derivatizedCM5 chip in flow cells at 15 microliters/min for a total volume of 50microliters. The amount of bound protein is determined during washing ofthe flow cell with BBS buffer (10mM HEPES, pH7.4, 150mM NaCl, 3.4 mMEDTA, 0.005% surfactant p20). Binding specificity for the protein ofinterest is determined by competition with soluble competitor in thepresence the protein of interest.

The flow cell surface can be regenerated by displacing bound protein bywashing with 20 microliters of 10 mM glycine-HCl, pH2.3. For kineticanalysis, the flow cells are tested at different flow rates anddifferent polypeptide densities on the CM5 chip. The on-rates andoff-rates can be determined using the kinetic evaluation program in aBIAevaluation 3 software.

Example 60 Virus Neutralization Assay

Antibodies of the invention may be assayed for their ability to inhibitor reduce ha ability of HIV-1 to infect (CCR5 expressing) cells using avirus neutralization assay such as the assay described in Zolla-Paznerand Sharpe, AIDS Res. Hum. Retrovir. 11:1449 (1995) which isincorporated in its entirety by reference herein. Briefly, 2×10⁵ restingPBMC are added to appropriate dilution(s) of antibodies of theinvention. After a one hour incubation, the cells are exposed to virusfor 2 hours, washed and resuspended in culture medium containing PHA andIL-2. At various time points after infection, such as at days 7 and 9,the amount of HBV p24 antigen in culture supernatant is measured usingELISA. The percent neutralization was calculated relative to a controlexperiment in which HIV was allow to infect cells in the absence ofantibodies of the invention, or alternatively (or in addition), in thepresence of an (isotype matched, if necessary) control antibody withirrelevant specificity.

A variation of this assay is to perform it on activated, rather thanresting, PBMC. This can be achieved by culturing the PBMC in thepresence of PHA and IL-2 for two days before performing the virusneutralization assay.

Example 61 MIP-1beta Binding Assay

Antibodies of the invention may be assayed for their ability to preventa natural ligand of CCR5, e.g., MIP1-beta, from binding to the CCR5receptor.

The following ¹²⁵-MIP1-beta binding assay is an example of one assaythat could be performed to determine the ability of an antibody of theinvention to prevent a natural ligand of CCR5, MIP1-beta, from bindingto the CCR5 receptor.

Twenty-five microCuries of ¹²⁵I-MIP-1beta (Amersham Pharmacia Biotech,Cat# fM310, 25 microCuries, 2000 Ci/mmol) is dissolved in 1 milliliterof distilled water to make a 12.5 nM stock solution. If cultured cells,such as CCR5CHO cells, are used in this experiment, they aretrypsinized, washed and resuspended at 10×10⁶ cells/milliliter inbinding buffer (1 mM CaCl₂, 5 mM MgCl₂, 50 mM Hepes, 0.5% BSA, 0.1%NaN₃, pH 7.5). If Peripheral Blood mononuclear cells (PBMC) are to beused in this assay, they are isolated from healthy donors andresuspended at 2×10⁶ cell/milliliter in binding buffer.

To determine what concentration or quantity of MIP-1beta would saturatethe cells in this assay, a series of ¹²⁵I-MIP-1beta dilutions at fourtimes the desired final concentration is made. (For example, for finalconcentrations of 3 nM, a 12 nM solution should be prepared). Typically,the desired final concentration of ¹²⁵I-MIP-1beta range from 3 nM downto 0.05 nM. Additionally, a solution of cold (non-radioactive) MIP-1betaat four times the desired final concentration is made. Typically, thedesired final concentration of cold MIP-1beta is 200 nM, so an 800 nMsolution is prepared.

To measure the total binding of 125-MIP-1beta to cells, 25 microlitersbinding buffer, 25 microliters hot 125I-MIP-1beta, and 50 microliterscell suspension are added to a U-bottom 96 well microplate (Costa, Cat#3799). The cells are always added last. If the binding of ¹²⁵I-MIP-1betais non-specific, it will not be effectively competed away by coldMIP-1beta. Therefore, to assess the specificity of binding, 25microliters cold MIP-1beta (800 nM), 25 microliters hot ¹²⁵I-MIP-1beta(various dilutions, and 50 microliters cell suspension are added to aU-bottom 96 well microplate. Again the cells are always added last. Themixtures are then incubated at room temperature in a shaker for onehour. After incubation, each sample is transferred to the top of tubescontaining 200 microliters of an oil mixture (2:1 dibutylphthalate:dioctyl phthalate). The tubes are spun at 12000 rpm for 20seconds using a microcentrifuge. The bottom of the tube, containing thecell pellet, is cut off and counted in a gamma counter. If the bindingof MIP-1beta is specific, less radioactivity will be measured in thegamma counter in the competition assay. As a control for ensuring thatthe MIP-1beta is binding, to G-protein Chemokine Receptor (CCR5), onemay choose to perform the experiment on a suitable CCR5 non-expressingcells, e.g., vector transfected CHO cells.

To perform a competition assay to determine if a chemokine or antibodycan compete with MIP-1 beta for binding to the same G-protein CoupledReceptor, a dilution series of cold chemokine or antibody at four timesthe desired final concentrations is prepared. Additionally, a solutionof ¹²⁵I-MIP-1beta at four times the desired final concentration isprepared. For this type of competition assay, a 2 nM solution of¹²⁵I-MIP-1beta, which will give a final concentration of 0.5 nM, isprepared.

To measure the total binding of ¹²⁵I-MIP-1beta to cells, 25 microlitersbinding buffer, 25 microliters hot ¹²⁵I-MIP-1beta (2 nM), and 50microliters cell suspension are added to a U-bottom 96 well microplate.The cells are always added last. If another substance (e.g.,anti-G-protein Chemokine Receptor (CCR5) antibody, or another chemokine)binds the receptor of MIP-1beta (i.e., G-protein Chemokine Receptor(CCR5)) the presence of increasing amounts of cold (non-radioactive)substance (e.g., anti-G-protein Chemokine Receptor (CCR5) antibody, oranother chemokine) will compete for binding to the MIP-1beta receptor(i.e., G-protein Chemokine Receptor (CCR5). Therefore, to determine if asubstance binds to the MIP-1beta receptor (i.e., G-protein ChemokineReceptor (CCR5), and inhibits (radioactively labelled) MIP-1beta frombinding to its receptor (i.e., G-protein Chemokine Receptor (CCR5), 25microliters of cold substance (e.g. anti-G-protein Chemokine Receptor(CCR5) antibody at various dilutions), 25 microliters hot ¹²⁵I-MIP-1beta(2 nM), and 50 microliters cell suspension are added to a U-bottom 96well microplate. Again the cells are always added last. The mixtures arethen incubated at room temperature in a shaker for one hour. Afterincubation, each sample is transferred to the top of tubes containing200 microliters of an oil:mixture (2:1 dibutyl phthalate:dioctylphthalate). The tubes are spun at 12000 rpm for 20 seconds using amicrocentrifuge. The bottom of the tube, containing the cell pellet, iscut off and counted in a gamma counter. If the binding of MIP-1beta isspecific, less radioactivity will be measured in the gamma counter inthe competition assay. As a control for ensuring that the MIP-1beta isbinding, to G-protein Chemokine Receptor (CCR5), one may choose toperform the experiment on a suitable CCR5 non-expressing cells, e.g.,vector transfected CHO cells.

An alternative assay, but similar, assay to determine the ability of anantibody of the invention to prevent a natural ligand of CCR5,MIP1-beta, from binding to the CCR5 receptor is described in Lopalco etal., J. Immunol., 164:3426 (2000) and in Trkola et al., Nature, 384:184(1996) which are incorporated in their entireties by reference herein.Briefly, 10⁶ CCR5 expressing cells (e.g., CD4+ T cells, CCR5transfected-CHO cells) are incubated on ice with appropriate dilution(s)of antibody of the invention. After 45 minutes of incubation, 0.2microCuries of radiolabelled MIP1-beta (e.g., 125I-MIP1-beta(DuPont-NEN, Boston, Mass.) is added to a final concentration of 0.1 nM.After a two hour incubation on ice, unbound radioactivity is removedusing a two step gradient, as described in Grassi et al., J Exp Med.174:53 (1991) which is incorporated in its entirety by reference herein,in which the lower layer consists of fetal calf serum containing 10%sucrose, and the upper layer consists of 80% silicone (Sigma Aldrich)and 20% mineral oil (Sigma Aldrich). Bound radioactivity in cell pelletsis measured in a gamma counter.

Example 62 Chemotaxis Assay

Polypeptides, and agonists or antagonists thereof of the invention maybe assayed for their ability to enhance, inhibit, or not significantlyalter chemotaxis of G-protein Chemokine Receptor (CCR5) expressing cellsin response to MIP1-beta. The G-protein Chemokine Receptor (CCR5)expressing cells may be a homogeneous population of purified G-proteinChemokine Receptor (CCR5) expressing cells or a heterogeneouspopulation, (e.g., peripheral blood mononuclear cells, PBMC).

The following assay to measure MIP1-beta induced chemotaxis of CCR5expressing cells involves labeling cells with a (fluorescent) tracermolecule, inducing chemotaxis in a well of a 96 well plate containing afilter through which cells can pass, and measuring the number ofmigrated cells via fluorescence emission. To perform this assay thefollowing materials are needed:

-   -   HBSS, without calcium, without magnesium (Biofluids Cat#:        p325-000)    -   Albumin, Bovine Powder, fraction V, IgG free (Sigma Cat#:        A-2058)    -   ChemoTx# 105-2 (for T cell, PBMC, NK cell), 108-1 (for        eosinophil, PMN) (Neuro Probe, Inc)    -   Calcein, AM (1 milligram/milliliter in dry DMSO) (Molecular        Probes Cat#: C-3099)    -   PBS, 1×, pH 7.4, without calcium and magnesium (Biofluids Cat#:        p312-00

Briefly, cells (e.g. PBMC) are washed twice with HBSS (Biofluids Cat#:p325-000)/0.1% BSA and resuspended in the buffer at 10×10⁶cells/milliliter. 5 microliters of calcein AM (1 milligram/milliliterstock) is added to 1 milliliter of the cell suspension. Cells areincubated at 37° C. incubator with loose cap for 30 minutes. Afterincubation, cells are washed twice with HBSS/0.1% BSA and resuspended at10×10⁶/milliliter in HBSS/0.1% BSA buffer. Twenty-nine microliters oftest chemokine or control buffer is added into bottom chamber of thechemotaxis microplate. The filter is snapped onto the 96-well plateposition making sure no air bubbles get between the filter and thesolution. Next, 20 microliters of the cells are loaded on top of thefilter and the plate is covered to prevent evaporation. The plate isthen incubated at 37° C. for 2 hours for T cells, PBMC, PMN and NK cellsand 3 hours for eosinophils. After incubating, carefully flush the topsurface of the filter with PBS buffer and then gently wipe thenon-migrated cells off the top of the filter with a squeegee. Read theplate and filter at Excitation of 485 nm/Emission 530 nm using CytoFluorfluorescence reader. Results are expressed as chemotactic index, whichrepresents the fold increase in the number of migrated cells in responseto chemokine over the spontaneous cell migration in control medium.

This protocol can easily be modified to assay if an agonist orantagonist of G-protein Chemokine Receptor (e.g., anti-G-proteinChemokine Receptor (CCR5) antibodies can enhance, inhibit or notsignificantly alter the ability of MIP-1beta to induce chemotaxis inCCR5 expressing cells. To do this, one might preincubate the cells withanti-G-protein Chemokine Receptor (CCR5) antibodies (possibly at severalconcentrations in order to generate a dose-response curve) prior toloading the cells on top of the filter.

An alternative assay to measure the ability of polypeptides, andagonists or antagonists thereof of the invention to enhance, inhibit, ornot significantly alter chemotaxis of G-protein Chemokine Receptor(CCR5) expressing cells in response to MIP-1beta can be performed in atranswell chamber, rather than in a 96 well microplate. To perform thisassay the following materials are needed:

-   -   RPMI-1640 (GIBCO-BRL Cat#: 21870-084)    -   Albumin, Bovine Powder, fraction V, IgG free (Sigma Cat#:        A-2058)    -   Transwell plate (Costar Cat#: 3421), 6.5 mm Diameter, 5.0 μm        pore size    -   MEP-1β (R&D Systems Cat#: 271-BME)    -   Lymphocyte Separation Medium (ICN Biochemical Cat #: 50494)

Briefly, PBMC are isolated from fresh human peripheral blood by usingLymphocyte Separation Medium and cultured in RPMI-1640 with 10% FBS for2 days. Cultured PBMC are resuspended in RPMI-1640/0.5% BSA at 20×10⁶cells/milliliter. MIP-1beta is diluted in RPMI-1640/0.5% BSA to finalconcentrations of 10, 100 and 1000 nanograms/milliliter. 600 microlitersof MIP-1beta solution or RPMI-1640/0.5% BSA alone is added to the bottomchamber of the transwell and 100 microliters of the cell suspension isadded to the top of the filter. Cells are incubated at 37° C. for 4hours. After the incubation, collect the cells that are migrated to thebottom the chamber and then perform a FACS analysis, for example, todetermine the number and type of the migrated cell population(s).Results are expressed as chemotactic index, which represents the foldincrease in the number of migrated cells in response to chemokine overthe spontaneous cell migration in control medium.

An additional assay to determine the ability of an antibody of theinvention to prevent a natural ligand of CCR5, MIP1-beta, from inducingchemotaxis in MIP-1beta expressing cells is described in Lopalco et al.,J. Immunol., 164:3426 (2000). Briefly PBMC are activated (e.g., withphytohemagglutinin and IL-2) for 3 days in the presence of antibodies ofthe invention. Then 3×10⁵ activated PBMC in 50 microliters of 1640 RPMIcontaining 3% human serum albumin are placed in the upper chamber of abare filter transwell with 5 micrometer pore size (CoStar). 1.5micrograms of MIP1-beta is plated in the lower chambers. Chemotaxis waspermitted to occur for one half hour while the transwell chamber wasincubated at 37 degrees Celsius. Cells that migrated from the upper tothe lower chamber were then quantified by FACS analysis. Results areexpressed in terms of a migration index, (i.e. the number of cellsmigrating to a lower chamber containing MIP1-beta/the number of cellsmigrating to a lower chamber containing only control medium.

Example 63 Calcium Mobilization Following Triggering of the G-ProteinChemokine Receptor (CCR5) Protein

When the G-protein Chemokine Receptor (CCR5) is triggered, calcium fromintracellular stores and from extracellular spaces is mobilized. Thiscalcium mobilization can be monitored using Fluorescent Ca⁺⁺ indicatorsthat can be excited with UV light, such as for example, Fura-2, AMavailable from Molecular Probes, Eugene, Oreg. (Cat# F-1221). An assayto monitor calcium mobilization using Fura-2 μM is described below.

Briefly, cells (e.g., purified PBMC or CCR5 transfected cells such asCCR5CHO cells) are suspended at 5×10⁶ cells/milliliter in calcium buffer(20 mM Hepes buffer, 125 mM NaCl, 5 mM KCl, 0.5 mM Glucose, 1 mM CaCl₂,1 mM MgCl₂, 0.025% BSA, pH 7.4). Fura-2, AM (50 μg/vial) is dissolved in25 μl of DMSO. Cells are labeled with dye by adding 1 μl of the Fura-2μM to 2 ml of cell suspension. The cells are then incubated for 30minutes at room temperature in the dark. After incubation, the cells arewashed twice with calcium buffer and suspended at 1×10⁶ cells/milliliterin calcium buffer. Two milliliters of the cell suspension is placed in acontinuously stirring cuvette at 37° C. [Ca⁺⁺]i concentration ismeasured using dual excitation wavelengths 340 nm and 380 nm, and asingle emission wavelength of 510 nm on an Hitachi spectrophotometer. Abaseline is established for 60 seconds before adding the test chemokine,or anti-G-protein coupled Receptor (CCR5) antibody. Twenty microlitersof the test chemokine (100 times the final concentration) is then addedto the cuvette and changes in intracellular calcium concentration aremonitored using the spectrophotometer. This assay can be used, forexample, if an anti-G-protein coupled Receptor (CCR5) antibody isagonistic (induces calcium mobilization) or antagonistic (fails toinduce calcium mobilization).

Example 64 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

Diabetic db+/db+ Mouse Model.

To demonstrate that G-protein Chemokine Receptor (CCR5) accelerates thehealing process, the genetically diabetic mouse model of wound healingis used. The full thickness wound healing model in the db+/db+ mouse isa well characterized, clinically relevant and reproducible model ofimpaired wound healing. Healing of the diabetic wound is dependent onformation of granulation tissue and re-epithelialization rather thancontraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992);Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Riflkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

G-protein Chemokine Receptor (CCR5) is administered using at a rangedifferent doses of G-protein Chemokine Receptor (CCR5), from 4 mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groupsreceived 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated; and 3) treatedgroup.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day I]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with G-protein Chemokine Receptor. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that G-protein Chemokine Receptor (CCR5 can acceleratethe healing process, the effects of multiple topical applications ofG-protein Chemokine Receptor (CCR5) on full thickness excisional skinwounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

G-protein Chemokine Receptor (CCR5) is administered using at a rangedifferent doses of G-protein Chemokine Receptor (CCR5), from 4 mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groupsreceived 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) G-protein Chemokine Receptor (CCR5) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day Iis 64 mm2, the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith G-protein Chemokine Receptor. A calibrated lens micrometer is usedby a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example test activity of G-proteinChemokine Receptor (CCR5) protein. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of G-proteinChemokine Receptor (CCR5) polynucleotides (e.g., gene therapy), agonists(including ligands), and/or antagonists of G-protein Chemokine Receptor.

Example 65 Evaluation of G-Protein Chemokine Receptor (CCR5) in aDiabetic Mouse Model

The diabetic mouse model used in Example 64 may also be used todetermine whether G-protein Chemokine Receptor (CCR5) is efficacious inpreventing, treating and/or ameliorating the diabetic condition per se.G-protein Chemokine Receptor (CCR5) is administered to db+/db+ miceparenterally for various periods of time either before or after the micehave developed diabetes, and blood glucose, and/or insulin levels, orother art-known methods for measuring disease severity, are measured todetermine whether administration prevents, slows, or lessens the onsetor severity of diabetes.

This example tests activity of G-protein Chemokine Receptor (CCR5)protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of G-protein Chemokine Receptor(CCR5) polynucleotides (e.g., gene therapy), agonists (includingligands), and/or antagonists of G-protein Chemokine Receptor.

Example 66 Evaluation of G-Protein Chemokine Receptor (CCR5) in a Modelof Inflammatory Bowel Disease & Colitis

The purpose of this study is to determine whether G-protein ChemokineReceptor (CCR5) is efficacious in a model of murine colitis induced byad libitum exposure to dextran sodium sulfate in the drinking water.

Six to eight week old female Swiss Webster mice (20-25 g, Charles River,Raleigh, N.C.)) are used in a model of inflammatory bowel diseaseinduced with a 4% solution of sodium sulfate (DSS, 36,000-44,000 MW,American International Chemistry, Natick, Mass.)) administered adlibitum for one week. Agonists, antagonists, preferably antibodies ofthe present invention, of G-protein Chemokine Receptor (CCR5) is givenby daily parenteral administration (n=10). Three parameters are used todetermine efficacy: 1) clinical score, based on evaluation of the stool;2) histological score, based on evaluation of the colon; and 3) weightchange. The clinical score are comprised of two parts totaling a maximumof score of four. Stool consistency is graded as: 0=firm; 1=loose; 2diarrhea. Blood in the stool is also evaluated on a 0 to 2 scale with0=no blood; 1=occult blood; and 2=gross rectal bleeding. A mean groupscore above 3 indicates probable lethality, and disease which hasprogressed beyond its treatable stage. Clinical scores are taken on Day0, 4, 5, 6, and 7. To arrive at a histological score, slides of theascending, transverse and descending colon are evaluated in a blindedfashion based on inflammation score (0-3) and crypt score (0-4). Bodyweight is measured daily. Data is expressed as mean +SEM. An unpairedStudent's t test is used to determine significant differences comparedto the disease control (* p<0.05; ** p<0.01; *** p<0.001).

Results from this study may suggest G-protein Chemokine Receptor (CCR5)role in IBD and colitis, including ulcerative colitis. Thus, agonists,antagonists, including antibodies of the present invention, andfragments of G-protein Chemokine Receptor (CCR5) may be used to treat,prevent, or ameliorate patients having IBD, colitis, and/or ulcerativecolitis, or any other inflammation of the intestine.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, Detailed.Description, and Examples is hereby incorporated herein by reference.The disclosure of U.S. application Ser. No. 09/195,662, filed Nov. 18,1998, is herein incorporated by reference. The disclosure of U.S.Provisional Application Nos. 60/181,258 filed Feb. 9, 2000; 60/187,999filed Mar. 9, 2000; and 60/234,336 filed Sep. 22, 2000 are hereinincorporated by reference. The disclosure of International PublicationWO 98/54317 is herein incorporated by reference. Additionally, thesequence listing of U.S. Pat. No. 5,707,815 is herein incorporated byreference.

1. An isolated antibody or fragment thereof comprising the amino acidsequence of the VH domain of the antibody expressed by the XF27/28.43E2hybridoma cell line deposited under ATCC Deposit Accession NumberPTA-4054 and the amino acid sequence of the VL domain of the antibodyexpressed by the XF27/28.43E2 hybridoma cell line deposited under ATCCDeposit Accession Number PTA-4054.
 2. The antibody or fragment thereofof claim 1 wherein the antibody or fragment thereof is selected from thegroup consisting of a whole immunoglobulin molecule, an scFv, a Fabfragment, an Fab′ fragment, an F(ab′)2, an Fv, and a disulfide linkedFv.
 3. The antibody or fragment thereof of claim 1 wherein the antibodyor fragment thereof is monoclonal.
 4. The antibody or fragment thereofof claim 1 wherein the antibody or fragment thereof is chimeric orhumanized.
 5. The antibody or fragment thereof of claim 1 whichcomprises a heavy chain immunoglobulin constant domain.
 6. The antibodyor fragment thereof of claim 5 wherein said heavy chain immunoglobulinconstant domain is selected from the group consisting of an IgM constantdomain, an IgG1 constant domain, an IgG2 constant domain, an IgG3constant domain, an IgG4 constant domain and an IgA constant domain. 7.The antibody or fragment thereof of claim 6 wherein said heavy chainimmunoglobulin constant domain is human.
 8. The antibody or fragmentthereof of claim 7 wherein said heavy chain immunoglobulin constantdomain is a human IgG4 constant domain.
 9. The antibody or fragmentthereof of claim 1 which comprises a light chain immunoglobulin constantdomain.
 10. The antibody or fragment thereof of claim 9 which comprisesa light chain immunoglobulin constant domain selected from the groupconsisting of a kappa constant domain and a lambda constant domain. 11.The antibody or fragment thereof of claim 10 wherein said light chainimmunoglobulin constant domain is human.
 12. The antibody or fragmentthereof of claim 1 which comprises a human IgG4 heavy chainimmunoglobulin constant domain and a human kappa chain immunoglobulinconstant domain.
 13. The antibody or fragment thereof of claim 1 whereinthe antibody or fragment thereof is coupled or conjugated to adetectable label.
 14. The antibody or fragment thereof of claim 13,wherein the detectable label is a radiolabel.
 15. The antibody orfragment thereof of claim 14, wherein the radiolabel is ¹²⁵I, ¹³¹I,¹¹¹In, ⁹⁰Y ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.
 16. The antibody or fragmentthereof of claim 13, wherein the detectable label is an enzyme, afluorescent label, a luminescent label, or a bioluminescent label. 17.The antibody or fragment thereof of claim 1 wherein the antibody orfragment thereof is biotinylated.
 18. The antibody or fragment thereofof claim 1 wherein the antibody or fragment thereof is attached to asolid support.
 19. The antibody or fragment thereof of claim 1 whereinthe antibody or fragment thereof inhibits the binding of HIV virus toCCR5 expressing cells.
 20. The antibody or fragment thereof of claim 1wherein the antibody or fragment thereof inhibits the ability of HIVvirus to infect CCR5 expressing cells.
 21. An isolated cell or cell linethat produces an antibody or fragment thereof comprising the amino acidsequence of the VH domain of the antibody expressed by the XF27/28.43E2hybridoma cell line deposited under ATCC Deposit Accession NumberPTA-4054 and the amino acid sequence of the VL domain of the antibodyexpressed by the XF27/28.43E2 hybridoma cell line deposited under ATCCDeposit Accession Number PTA-4054.
 22. The XF27/28.43E2 hybridoma cellline deposited under ATCC Deposit Accession Number PTA-4054.
 23. Anantibody produced by the XF27/28.43E2 hybridoma cell line depositedunder ATCC Deposit Accession Number PTA-4054.
 24. An isolated antibodyor fragment thereof comprising the amino acid sequence of the VHCDR1,VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of the antibody expressed bythe XF27/28.43E2 hybridoma cell line deposited under ATCC DepositAccession Number PTA-4054, wherein said antibody or fragment thereofspecifically binds CCR5.
 25. The antibody or fragment thereof of claim24 that binds a CCR5 polypeptide purified from a cell culture whereinsaid CCR5 polypeptide is encoded by a polynucleotide encoding aminoacids 1 to 352 of SEQ ID NO:22.
 26. The antibody or fragment thereof ofclaim 24 which is a human antibody or fragment thereof.
 27. An isolatedhuman antibody or fragment thereof comprising an amino acid sequencethat is at least 85% identical to the VH domain of the antibodyexpressed by the XF27/28.43E2 hybridoma cell line deposited under ATCCDeposit Accession Number PTA-4054 and an amino acid sequence that is atleast 85% identical to the VL domain of the antibody expressed by theXF27/28.43E2 hybridoma cell line deposited under ATCC Deposit AccessionNumber PTA-4054; wherein said antibody or fragment thereof specificallybinds a CCR5 polypeptide.
 28. The antibody or fragment thereof of claim27 comprising an amino acid sequence that is at least 90% identical tothe VH domain of the antibody expressed by the XF27/28.43E2 hybridomacell line deposited under ATCC Deposit Accession Number PTA-4054 and anamino acid sequence that is at least 90% identical to the VL domain ofthe antibody expressed by the XF27/28.43E2 hybridoma cell line depositedunder ATCC Deposit Accession Number PTA-4054.
 29. The antibody orfragment thereof of claim 27 comprising an amino acid sequence that isat least 95% identical to the VH domain of the antibody expressed by theXF27/28.43E2 hybridoma cell line deposited under ATCC Deposit AccessionNumber PTA-4054 and an amino acid sequence that is at least 95%identical to the VL domain of the antibody expressed by the XF27/28.43E2hybridoma cell line deposited under ATCC Deposit Accession NumberPTA-4054.
 30. The antibody or fragment thereof of claim 27 comprisingthe amino acid sequence of the VH domain of the antibody expressed bythe XF27/28.43E2 hybridoma cell line deposited under ATCC DepositAccession Number PTA-4054; and the amino acid sequence of the VL domainof the antibody expressed by the XF27/28.43E2 hybridoma cell linedeposited under ATCC Deposit Accession Number PTA-4054.
 31. The antibodyor fragment thereof of claim 27 wherein said CCR5 polypeptide isexpressed on the surface of a cell and wherein said CCR5 polypeptide isencoded by a polynucleotide encoding amino acids 1 to 352 of SEQ IDNO:22.
 32. The antibody or fragment thereof of claim 31 which binds tothe second extracellular loop of said CCR5 polypeptide.
 33. The antibodyor fragment thereof of claim 27 wherein the antibody or fragment thereofis selected from the group consisting of a whole immunoglobulinmolecule, an scFv, a Fab fragment, an Fab′ fragment, an F(ab′)2, an Fv,and a disulfide linked Fv.
 34. The antibody or fragment thereof of claim27 wherein the antibody or fragment thereof is monoclonal.
 35. Theantibody or fragment thereof of claim 27 wherein the antibody orfragment thereof is human.
 36. The antibody or fragment thereof of claim27 wherein the antibody or fragment thereof is chimeric or humanized.37. The antibody or fragment thereof of claim 27 which comprises a heavychain immunoglobulin constant domain.
 38. The antibody or fragmentthereof of claim 37 wherein said heavy chain immunoglobulin constantdomain is selected from the group consisting of an IgM constant domain,an IgG1 constant domain, an IgG2 constant domain, an IgG3 constantdomain, an IgG4 constant domain and an IgA constant domain.
 39. Theantibody or fragment thereof of claim 38 wherein said heavy chainimmunoglobulin constant domain is human.
 40. The antibody or fragmentthereof of claim 27 which comprises a light chain immunoglobulinconstant domain.
 41. The antibody or fragment thereof of claim 40 whichcomprises a light chain immunoglobulin constant domain selected from thegroup consisting of a kappa constant domain and a lambda constantdomain.
 42. The antibody or fragment thereof of claim 41 wherein saidlight chain immunoglobulin constant domain is human.
 43. The antibody orfragment thereof of claim 27 wherein the antibody or fragment thereofhas a dissociation constant (K_(D)) of less than or equal to 10⁻⁹M. 44.The antibody or fragment thereof of claim 27 wherein the antibody orfragment thereof is coupled or conjugated to a detectable label.
 45. Theantibody or fragment thereof of claim 44, wherein the detectable labelis a radiolabel.
 46. The antibody or fragment thereof of claim 45,wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or¹⁵³Sm.
 47. The antibody or fragment thereof of claim 46, wherein thedetectable label is an enzyme, a fluorescent label, a luminescent label,or a bioluminescent label.
 48. The antibody or fragment thereof of claim27 wherein the antibody or fragment thereof is biotinylated.
 49. Theantibody or fragment thereof of claim 27 wherein the antibody orfragment thereof is attached to a solid support.
 50. The antibody orfragment thereof of claim 27 wherein the antibody or fragment thereofspecifically binds a CCR5 polypeptide in a Western blot.
 51. Theantibody or fragment thereof of claim 27 wherein the antibody orfragment thereof specifically binds a CCR5 polypeptide in an ELISA. 52.The antibody or fragment thereof of claim 27 wherein the antibody orfragment thereof is an antagonist of CCR5.
 53. The antibody or fragmentthereof of claim 27 wherein the antibody or fragment thereof inhibitsthe binding of MIP-1beta to CCR5.
 54. The antibody or fragment thereofof claim 27 wherein the antibody or fragment thereof inhibits thebinding of Eotaxin, RANTES, MCP-1, MCP-2, MCP-3 or MIP-1 alpha to CCR5.55. The antibody or fragment thereof of claim 27 wherein the antibody orfragment thereof inhibits chemotaxis of CCR5 expressing cells whereinsaid chemotaxis is induced by MIP-1beta.
 56. The antibody or fragmentthereof of claim 27 wherein the antibody or fragment thereof inhibitschemotaxis of CCR5 expressing cells wherein said chemotaxis is inducedby Eotaxin, RANTES, MCP-1, MCP-2, MCP-3 or MIP-1 alpha.
 57. The antibodyor fragment thereof of claim 27 wherein the antibody or fragment thereofdownregulates CCR5 expression on CCR5 expressing cells.
 58. The antibodyor fragment thereof of claim 27 wherein the antibody or fragment thereofinhibits the binding of HIV virus to CCR5 expressing cells.
 59. Theantibody or fragment thereof of claim 27 wherein the antibody orfragment thereof inhibits the ability of HIV virus to infect CCR5expressing cells.
 60. An isolated cell that produces a human antibody orfragment thereof comprising an amino acid sequence that is at least 85%identical to the VH domain of the antibody expressed by the XF27/28.43E2hybridoma cell line deposited under ATCC Deposit Accession NumberPTA-4054 and an amino acid sequence that is at least 85% identical tothe VL domain of the antibody expressed by the XF27/28.43E2 hybridomacell line deposited under ATCC Deposit Accession Number PTA-4054;wherein said antibody or fragment thereof specifically binds a CCR5polypeptide.